A novel iron chaperone complex delivers iron to the cytosolic [2Fe-2S] cluster distribution system
Ontology highlight
ABSTRACT:
INSTRUMENT(S): LTQ Orbitrap XL
ORGANISM(S): Homo Sapiens (ncbitaxon:9606)
SUBMITTER: 
James Wohlschlegel
PROVIDER: MSV000083887 | MassIVE | Tue Jun 04 08:24:00 BST 2019
REPOSITORIES: MassIVE
Similar Datasets
Project description:PCBP1 and control immunoprecipitates (IP) isolated from HEK293 cell lines treated with ferric ammonium citrate (FAC) or desferal (DFO) were analyzed by LC-MS. Each IP was fractionated by SCX into four fractions and then individually analyzed using online reversed phase chromatography followed by MS/MS.
2019-06-04 | MSV000083887 | GNPS
Project description:Hundreds of cellular proteins require iron cofactors for activity, and cells express systems for their assembly and distribution. Molecular details of the cytosolic iron pool used for iron cofactors are lacking, but iron chaperones of the poly(rC)-binding protein (PCBP) family play a key role in ferrous ion distribution. Here we show that, in cells and in vitro, PCBP1 coordinates iron via conserved cysteine and glutamate residues and a molecule of noncovalently bound glutathione (GSH). Proteomics analysis of PCBP1-interacting proteins identified BolA2, which functions, in complex with Glrx3, as a cytosolic [2Fe-2S] cluster chaperone. The Fe-GSH-bound form of PCBP1 complexes with cytosolic BolA2 via a bridging Fe ligand. Biochemical analysis of PCBP1 and BolA2, in cells and in vitro, indicates that PCBP1-Fe-GSH-BolA2 serves as an intermediate complex required for the assembly of [2Fe-2S] clusters on BolA2-Glrx3, thereby linking the ferrous iron and Fe-S distribution systems in cells.
| S-EPMC6702080 | biostudies-literature
Project description:Cells contain hundreds of proteins that require iron cofactors for activity. Iron cofactors are synthesized in the cell, but the pathways involved in distributing heme, iron-sulfur clusters, and ferrous/ferric ions to apoproteins remain incompletely defined. In particular, cytosolic monothiol glutaredoxins and BolA-like proteins have been identified as [2Fe-2S]-coordinating complexes in vitro and iron-regulatory proteins in fungi, but it is not clear how these proteins function in mammalian systems or how this complex might affect Fe-S proteins or the cytosolic Fe-S assembly machinery. To explore these questions, we use quantitative immunoprecipitation and live cell proximity-dependent biotinylation to monitor interactions between Glrx3, BolA2, and components of the cytosolic iron-sulfur cluster assembly system. We characterize cytosolic Glrx3·BolA2 as a [2Fe-2S] chaperone complex in human cells. Unlike complexes formed by fungal orthologs, human Glrx3-BolA2 interaction required the coordination of Fe-S clusters, whereas Glrx3 homodimer formation did not. Cellular Glrx3·BolA2 complexes increased 6-8-fold in response to increasing iron, forming a rapidly expandable pool of Fe-S clusters. Fe-S coordination by Glrx3·BolA2 did not depend on Ciapin1 or Ciao1, proteins that bind Glrx3 and are involved in cytosolic Fe-S cluster assembly and distribution. Instead, Glrx3 and BolA2 bound and facilitated Fe-S incorporation into Ciapin1, a [2Fe-2S] protein functioning early in the cytosolic Fe-S assembly pathway. Thus, Glrx3·BolA is a [2Fe-2S] chaperone complex capable of transferring [2Fe-2S] clusters to apoproteins in human cells.
| S-EPMC5077177 | biostudies-literature
Project description:Ferritins are the main iron storage proteins found in animals, plants, and bacteria. The capacity to store iron in ferritin is essential for life in mammals, but the mechanism by which cytosolic iron is delivered to ferritin is unknown. Human ferritins expressed in yeast contain little iron. Human poly (rC)-binding protein 1 (PCBP1) increased the amount of iron loaded into ferritin when expressed in yeast. PCBP1 bound to ferritin in vivo and bound iron and facilitated iron loading into ferritin in vitro. Depletion of PCBP1 in human cells inhibited ferritin iron loading and increased cytosolic iron pools. Thus, PCBP1 can function as a cytosolic iron chaperone in the delivery of iron to ferritin.
| S-EPMC2505357 | biostudies-literature
Project description:Cytosolic iron-sulfur (Fe-S) cluster assembly (CIA) pathway delivers Fe-S clusters to nuclear and cytosolic Fe-S proteins involved in essential cellular functions. This delivery process is regulated by availability of iron and oxygen, but it remains unclear how CIA components orchestrate the cluster transfer under varying cellular environments. Here, we investigated the organization of CIA machinery under various conditions. We developed a targeted proteomics assay monitoring known CIA factors. Using this assay, we were able to detect NUBP1, CIAO3 and CIA substrates in immunoprecipitates of NUBP2, a component of the CIA scaffold complex. We also revealed that NUBP2 transiently associates with the CIA targeting complex (MMS19, CIAO1, CIAO2B), indicating the possible existence of CIA metabolons. We observed stronger interactions between CIAO3 and the CIA scaffold complex upon iron supplementation or low oxygen tension, while iron chelation and reactive oxygen species weaken CIAO3 interactions with CIA components. We further demonstrated that CIAO3 mutant with defective Fe-S cluster binding failed to integrate into the metabolons. However, these mutants unexpectedly exhibit stronger association with CIA substrates regardless of their reduced association with the CIA targeting complex, implicating that CIAO3 and CIA substrates may present in complexes independent of the CIA targeting complex. Together, our data suggested that CIA components potentially form metabolons whose assembly are regulated by environmental cues and require Fe-S cluster incorporation in CIAO3. These findings provided additional evidence that the CIA pathway adapts to changes in cellular environment through complex reorganization.
2021-11-16 | MSV000088394 | MassIVE
Project description:The cytosolic iron-sulfur (Fe-S) cluster assembly (CIA) pathway delivers Fe-S clusters to nuclear and cytosolic Fe-S proteins involved in essential cellular functions. Although the delivery process is regulated by the availability of iron and oxygen, it remains unclear how CIA components orchestrate the cluster transfer under varying cellular environments. Here, we utilized a targeted proteomics assay for monitoring CIA factors and substrates to characterize the CIA machinery. We find that nucleotide-binding protein 1 (NUBP1/NBP35), cytosolic iron-sulfur assembly component 3 (CIAO3/NARFL), and CIA substrates associate with nucleotide-binding protein 2 (NUBP2/CFD1), a component of the CIA scaffold complex. NUBP2 also weakly associates with the CIA targeting complex (MMS19, CIAO1, and CIAO2B) indicating the possible existence of a higher order complex. Interactions between CIAO3 and the CIA scaffold complex are strengthened upon iron supplementation or low oxygen tension, while iron chelation and reactive oxygen species weaken CIAO3 interactions with CIA components. We further demonstrate that CIAO3 mutants defective in Fe-S cluster binding fail to integrate into the higher order complexes. However, these mutants exhibit stronger associations with CIA substrates under conditions in which the association with the CIA targeting complex is reduced suggesting that CIAO3 and CIA substrates may associate in complexes independently of the CIA targeting complex. Together, our data suggest that CIA components potentially form a metabolon whose assembly is regulated by environmental cues and requires Fe-S cluster incorporation in CIAO3. These findings provide additional evidence that the CIA pathway adapts to changes in cellular environment through complex reorganization.
| S-EPMC9243173 | biostudies-literature
Project description:An ordinary differential equation-based mathematical model was developed to describe trafficking and regulation of iron in growing fermenting budding yeast. Accordingly, environmental iron enters the cytosol and moves into mitochondria and vacuoles. Dilution caused by increasing cell volume is included. Four sites are regulated, including those in which iron is imported into the cytosol, mitochondria, and vacuoles, and the site at which vacuolar Fe(II) is oxidized to Fe(III). The objective of this study was to determine whether cytosolic iron (Fecyt) and/or a putative sulfur-based product of iron-sulfur cluster (ISC) activity was/were being sensed in regulation. The model assumes that the matrix of healthy mitochondria is anaerobic, and that in ISC mutants, O2 diffuses into the matrix where it reacts with nonheme high spin Fe(II) ions, oxidizing them to nanoparticles and generating reactive oxygen species. This reactivity causes a further decline in ISC/heme biosynthesis, which ultimately gives rise to the diseased state. The ordinary differential equations that define this model were numerically integrated, and concentrations of each component were plotted versus the concentration of iron in the growth medium and versus the rate of ISC/heme biosynthesis. Model parameters were optimized by fitting simulations to literature data. The model variant that assumed that both Fecyt and ISC biosynthesis activity were sensed in regulation mimicked observed behavior best. Such "dual sensing" probably arises in real cells because regulation involves assembly of an ISC on a cytosolic protein using Fecyt and a sulfur species generated in mitochondria during ISC biosynthesis and exported into the cytosol.
| S-EPMC4646409 | biostudies-literature
Project description:Human cytosolic monothiol glutaredoxin-3 (GLRX3) is a protein essential for the maturation of cytosolic [4Fe-4S] proteins. We show here that dimeric cluster-bridged GLRX3 transfers its [2Fe-2S]2+ clusters to the human P-loop NTPase NUBP1, an essential early component of the cytosolic iron-sulfur assembly (CIA) machinery. Specifically, we observed that [2Fe-2S]2+ clusters are transferred from GLRX3 to monomeric apo NUBP1 and reductively coupled to form [4Fe-4S]2+ clusters on both N-terminal CX13CX2CX5C and C-terminal CPXC motifs of NUBP1 in the presence of glutathione that acts as a reductant. In this process, cluster binding to the C-terminal motif of NUBP1 promotes protein dimerization, while cluster binding to the N-terminal motif does not affect the quaternary structure of NUBP1. The cluster transfer/assembly process is not complete on both N- and C-terminal motifs and indeed requires a reductant stronger than GSH to increase its efficiency. We also showed that the [4Fe-4S]2+ cluster formed at the N-terminal motif of NUBP1 is tightly bound, while the [4Fe-4S]2+ cluster bound at the C-terminal motif is labile. Our findings provide the first evidence for GLRX3 acting as a [2Fe-2S] cluster chaperone in the early stage of the CIA machinery.
| S-EPMC8007109 | biostudies-literature
Project description:Frataxin, a mitochondrial protein that is directly involved in regulating cellular iron homeostasis, has been suggested to serve as an iron chaperone during cellular Fe-S cluster biosynthesis. In humans, decreased amounts or impaired function of frataxin causes the autosomal recessive neurodegenerative disorder Friedreich's ataxia. Cellular production of Fe-S clusters is accomplished by the Fe cofactor assembly platform enzymes Isu (eukaryotes) and IscU (prokaryotes). In this report, we have characterized the overall stability and iron binding properties of the Drosophila frataxin homologue (Dfh). Dfh is highly folded with secondary structural elements consistent with the structurally characterized frataxin orthologs. While the melting temperature ( T M approximately 59 degrees C) and chemical stability ([urea] 50% approximately 2.4 M) of Drosophila frataxin, measured using circular dichroism (CD) and fluorescence spectroscopy, closely match values determined for the human ortholog, pure Dfh is more stable against autodegradation than both the human and yeast proteins. The ferrous iron binding affinity ( K d approximately 6.0 microM) and optimal metal to protein stoichiometry (1:1) for Dfh have been measured using isothermal titration calorimetry (ITC). Under anaerobic conditions with salt present, holo-Dfh is a stable iron-loaded protein monomer. Frataxin prevents reactive oxygen species-induced oxidative damage to DNA when presented with both Fe(II) and H 2O 2. Ferrous iron bound to Dfh is high-spin and held in a partially symmetric Fe-(O/N) 6 coordination environment, as determined by X-ray absorption spectroscopy (XAS). Extended X-ray absorption fine structure (EXAFS) simulations indicate the average Fe-O/N bond length in Dfh is 2.13 A, consistent with a ligand geometry constructed by water and carboxylate oxygens most likely supplied in part by surface-exposed conserved acidic residues located on helix 1 and strand 1 in the structurally characterized frataxin orthologs. The iron-dependent binding affinity ( K d approximately 0.21 microM) and optimal holo-Dfh to Isu monomer stoichiometry (1:1) have also been determined using ITC. Finally, frataxin mediates the delivery of Fe(II) to Isu, promoting Fe-S cluster assembly in vitro. The Dfh-assisted assembly of Fe-S clusters occurs with an observed kinetic rate constant ( k obs) of 0.096 min (-1).
| S-EPMC2664653 | biostudies-literature