Self-assembly Is Prerequisite for Catalysis of Fe(II) Oxidation by Catalytically Active Subunits of Ferritin

Ebrahimi, Kourosh Honarmand; Hagedoorn, Peter‐Leon; Hagen, Wilfred R.

doi:10.1074/jbc.m115.678375

Cited by 18 publications

(14 citation statements)

References 55 publications

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“…The hydrodynamic diameters measured by DLS experiments were 5.8 nm for the dissociated state in the absence of MgCl 2 , in agreement with the predicted theoretical value for a dimer, 33 and approximately 14 nm for the associated state in 20 mM MgCl 2 ( Fig. S2 †).…”

Section: Ferritin's Assembly Assessment By Dlssupporting

confidence: 86%

Excimer based fluorescent pyrene–ferritin conjugate for protein oligomerization studies and imaging in living cells

Benni

Trabuco²,

Stasio

et al. 2018

RSC Adv.

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Ferritin self-assembly has been widely exploited for the synthesis of a variety of nanoparticles for drugdelivery and diagnostic applications. However, despite the crucial role of ferritin self-assembly mechanism for probes encapsulation, little is known about the principles behind the oligomerization mechanism. In the present work, the novel "humanized" chimeric Archaeal ferritin HumAfFt, displaying the transferrin receptor-1 (TfR1) recognition motif typical of human H homopolymer and the unique salttriggered oligomerization properties of Archaeoglobus fulgidus ferritin (AfFt), was site-selectively labeled with N-(1-pyrenyl)maleimide on a topologically selected cysteine residue inside the protein cavity, next to the dimer interface. Pyrene characteristic fluorescence features were exploited to investigate the transition from a dimeric to a cage-like 24-meric state and to visualize the protein in vitro by two photon fluorescence microscopy. Indeed, pyrene fluorescence changes upon ferritin self-assembly allowed to establish, for the first time, the kinetic and thermodynamic details of the archaeal ferritins oligomerization mechanism. In particular, the magnesium induced oligomerization proved to be faster than the monovalent cation-triggered process, highly cooperative, complete at low MgCl 2 concentrations, and reversed by treatment with EDTA. Moreover, pyrene intense excimer fluorescence was successfully visualized in vitro by two photon fluorescence microscopy as pyrene-labeled HumAfFt was actively uptaken into HeLa cells by human transferrin receptor TfR1 recognition, thus representing a unique nano-device building block for two photon fluorescence cell imaging.

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Section: Ferritin's Assembly Assessment By Dlssupporting

confidence: 86%

Excimer based fluorescent pyrene–ferritin conjugate for protein oligomerization studies and imaging in living cells

Benni

Trabuco²,

Stasio

et al. 2018

RSC Adv.

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“…Mass spectrometry was performed using a protocol derived from [ 36 ] and similar to [ 37 ]. Briefly explained, full scan MS spectra (from m/z 400–1500, charge states 2 and higher) were acquired at a resolution of 30,000 at m/z 400 after accumulation to a target value of 10 6 ions (automatic gain control).…”

Section: Methodsmentioning

confidence: 99%

Membrane potential independent transport of NH3 in the absence of ammonium permeases in Saccharomyces cerevisiae

et al. 2017

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BackgroundMicrobial production of nitrogen containing compounds requires a high uptake flux and assimilation of the N-source (commonly ammonium), which is generally coupled with ATP consumption and negatively influences the product yield. In the industrial workhorse Saccharomyces cerevisiae, ammonium (NH4 +) uptake is facilitated by ammonium permeases (Mep1, Mep2 and Mep3), which transport the NH4 + ion, resulting in ATP expenditure to maintain the intracellular charge balance and pH by proton export using the plasma membrane-bound H+-ATPase.ResultsTo decrease the ATP costs for nitrogen assimilation, the Mep genes were removed, resulting in a strain unable to uptake the NH4 + ion. Subsequent analysis revealed that growth of this ∆mep strain was dependent on the extracellular NH3 concentrations. Metabolomic analysis revealed a significantly higher intracellular NHX concentration (3.3-fold) in the ∆mep strain than in the reference strain. Further proteomic analysis revealed significant up-regulation of vacuolar proteases and genes involved in various stress responses.ConclusionsOur results suggest that the uncharged species, NH3, is able to diffuse into the cell. The measured intracellular/extracellular NHX ratios under aerobic nitrogen-limiting conditions were consistent with this hypothesis when NHx compartmentalization was considered. On the other hand, proteomic analysis indicated a more pronounced N-starvation stress response in the ∆mep strain than in the reference strain, which suggests that the lower biomass yield of the ∆mep strain was related to higher turnover rates of biomass components.Electronic supplementary materialThe online version of this article (doi:10.1186/s12918-016-0381-1) contains supplementary material, which is available to authorized users.

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“…%A II B II C 0 = (Y Â 100) À %A II B II C II (8) %A II B 0 C 0 = (Z Â 100) À %A II B 0 C II (9) in which %Fe(II) in site B or C is obtained from the results of Mössbauer spectroscopy for samples before the addition of dioxygen. Using eqn (6)- (9) we found that the percentage of (A II B II C 0 ) subunits in PfFtn and HuHF is circa 52% and 42%, respectively, and that of (A II B II C II ) subunits in PfFtn and HuHF is 32% and 14%, respectively (Fig. 3).…”

Section: Paper Molecular Biosystemsmentioning

confidence: 99%

Spectroscopic evidence for the role of a site of the di-iron catalytic center of ferritins in tuning the kinetics of Fe(ii) oxidation

et al. 2016

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Ferritin is a nanocage protein made of 24 subunits. Its major role is to manage intracellular concentrations of free Fe(ii) and Fe(iii) ions, which is pivotal for iron homeostasis across all domains of life. This function of the protein is regulated by a conserved di-iron catalytic center and has been the subject of extensive studies over the past 50 years. Yet, it has not been fully understood how Fe(ii) is oxidized in the di-iron catalytic center and it is not known why eukaryotic and microbial ferritins oxidize Fe(ii) with different kinetics. In an attempt to obtain a new insight into the mechanism of Fe(ii) oxidation and understand the origin of the observed differences in the catalysis of Fe(ii) oxidation among ferritins we studied and compared the mechanism of Fe(ii) oxidation in the eukaryotic human H-type ferritin (HuHF) and the archaeal ferritin from Pyrococcus furiosus (PfFtn). The results show that the spectroscopic characteristics of the intermediate of Fe(ii) oxidation and the Fe(iii)-products are the same in these two ferritins supporting the proposal of unity in the mechanism of Fe(ii) oxidation among eukaryotic and microbial ferritins. Moreover, we observed that a site in the di-iron catalytic center controls the distribution of Fe(ii) among subunits of HuHF and PfFtn differently. This observation explains the reported differences between HuHF and PfFtn in the kinetics of Fe(ii) oxidation and the amount of O consumed per Fe(ii) oxidized. These results provide a fresh understanding of the mechanism of Fe(ii) oxidation by ferritins.

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Self-assembly Is Prerequisite for Catalysis of Fe(II) Oxidation by Catalytically Active Subunits of Ferritin

Cited by 18 publications

References 55 publications

Excimer based fluorescent pyrene–ferritin conjugate for protein oligomerization studies and imaging in living cells

Excimer based fluorescent pyrene–ferritin conjugate for protein oligomerization studies and imaging in living cells

Membrane potential independent transport of NH3 in the absence of ammonium permeases in Saccharomyces cerevisiae

Spectroscopic evidence for the role of a site of the di-iron catalytic center of ferritins in tuning the kinetics of Fe(ii) oxidation

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