NEET proteins are conserved 2Fe-2S proteins that regulate the levels of iron and reactive oxygen species in plant and mammalian cells. Previous studies of seedlings with constitutive expression of AtNEET, or its dominant-negative variant H89C (impaired in 2Fe-2S cluster transfer), revealed that disrupting AtNEET function causes oxidative stress, chloroplast iron overload, activation of iron-deficiency responses, and cell death. Because disrupting AtNEET function is deleterious to plants, we developed an inducible expression system to study AtNEET function in mature plants using a time-course proteomics approach. Here, we report that the suppression of AtNEET cluster transfer function results in drastic changes in the expression of different members of the ferredoxin (Fd), Fd-thioredoxin (TRX) reductase (FTR), and TRX network of Arabidopsis, as well as in cytosolic cluster assembly proteins. In addition, the expression of Yellow Stripe-Like 6 (YSL6), involved in iron export from chloroplasts was elevated. Taken together, our findings reveal new roles for AtNEET in supporting the Fd-TFR-TRX network of plants, iron mobilization from the chloroplast, and cytosolic 2Fe-2S cluster assembly. In addition, we show that the AtNEET function is linked to the expression of glutathione peroxidases (GPXs), which play a key role in the regulation of ferroptosis and redox balance in different organisms.
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A VDAC1-mediated NEET protein chain transfers [2Fe-2S] clusters between the mitochondria and the cytosol and impacts mitochondrial dynamics
Mitochondrial inner NEET (MiNT) and the outer mitochondrial membrane (OMM) mitoNEET (mNT) proteins belong to the NEET protein family. This family plays a key role in mitochondrial labile iron and reactive oxygen species (ROS) homeostasis. NEET proteins contain labile [2Fe-2S] clusters which can be transferred to apo-acceptor proteins. In eukaryotes, the biogenesis of [2Fe-2S] clusters occurs within the mitochondria by the iron–sulfur cluster (ISC) system; the clusters are then transferred to [2Fe-2S] proteins within the mitochondria or exported to cytosolic proteins and the cytosolic iron–sulfur cluster assembly (CIA) system. The last step of export of the [2Fe-2S] is not yet fully characterized. Here we show that MiNT interacts with voltage-dependent anion channel 1 (VDAC1), a major OMM protein that connects the intermembrane space with the cytosol and participates in regulating the levels of different ions including mitochondrial labile iron (mLI). We further show that VDAC1 is mediating the interaction between MiNT and mNT, in which MiNT transfers its [2Fe-2S] clusters from inside the mitochondria to mNT that is facing the cytosol. This MiNT–VDAC1–mNT interaction is shown both experimentally and by computational calculations. Additionally, we show that modifying MiNT expression in breast cancer cells affects the dynamics of mitochondrial structure and morphology, mitochondrial function, and breast cancer tumor growth. Our findings reveal a pathway for the transfer of [2Fe-2S] clusters, which are assembled inside the mitochondria, to the cytosol.
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- NSF-PAR ID:
- 10378745
- Date Published:
- Journal Name:
- Proceedings of the National Academy of Sciences
- Volume:
- 119
- Issue:
- 7
- ISSN:
- 0027-8424
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Peptides coordinated to iron–sulfur clusters, referred to as maquettes, represent a synthetic strategy for constructing biomimetic models of iron–sulfur metalloproteins. These maquettes have been successfully employed as building blocks of engineered heme‐containing proteins with electron‐transfer functionality; however, they have yet to be explored in reactivity studies. The concept of iron–sulfur nesting in peptides is a leading hypothesis in Origins‐of‐Life research as a plausible path to bridge the discontinuity between prebiotic chemical transformations and extant enzyme catalysis. Based on past biomimetic and biochemical research, we put forward a mechanism of maquette reconstitution that guides our development of computational tools and methodologies. In this study, we examined a key feature of the first stage of maquette formation, which is the secondary structure of aqueous peptide models using molecular dynamics simulations based on the AMBER99SB empirical force field. We compared and contrasted S…S distances, [2Fe‐2S] and [4Fe‐4S] nests, and peptide conformations via Ramachandran plots for dissolved Cys and Gly amino acids, the CGGCGGC 7‐mer, and the GGCGGGCGGCGGW 16‐mer peptide. Analytical tools were developed for following the evolution of secondary structural features related to [Fe‐S] cluster nesting along 100 ns trajectories. Simulations demonstrated the omnipresence of peptide nests for preformed [2Fe‐2S] clusters; however, [4Fe‐4S] cluster nests were observed only for the 16‐mer peptide with lifetimes of a few nanoseconds. The origin of the [4Fe‐4S] nest and its stability was linked to a “kinked‐ribbon” peptide conformation. Our computational approach lays the foundation for transitioning into subsequent stages of maquette reconstitution, those being the formation of iron ion/iron–sulfur coordinated peptides. © 2018 Wiley Periodicals, Inc.
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It was previously postulated that when intracellular free iron content is elevated in bacteria, the Ferric uptake regulator (Fur) binds its co-repressor a mononuclear ferrous iron to regulate intracellular iron homeostasis. However, the proposed iron-bound Fur had not been identified in any bacteria. In previous studies, we have demonstrated that Escherichia coli Fur binds a [2Fe-2S] cluster in response to elevation of intracellular free iron content, and that binding of the [2Fe-2S] cluster turns on Fur as an active repressor to bind a specific DNA sequence known as the Fur-box. Here we find that the iron-sulfur cluster assembly scaffold protein IscU is required for the [2Fe-2S] cluster assembly in Fur, as deletion of IscU inhibits the [2Fe-2S] cluster assembly in Fur and prevents activation of Fur as a repressor in E. coli cells in response to elevation of intracellular free iron content. Additional studies reveal that IscU promotes the [2Fe-2S] cluster assembly in apo-form Fur and restores its Fur-box binding activity in vitro. While IscU is also required for the [2Fe-2S] cluster assembly in the Haemophilus influenzae Fur in E. coli cells, deletion of IscU does not significantly affect the [2Fe-2S] cluster assembly in the E. coli ferredoxin and siderophore-reductase FhuF. Our results suggest that IscU may have a unique role for the [2Fe-2S] cluster assembly in Fur, and that regulation of intracellular iron homeostasis is closely coupled with iron-sulfur cluster biogenesis in E. coli.more » « less
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Abstract Iron-sulfur clusters are essential for life and defects in their biosynthesis lead to human diseases. The mechanism of cluster assembly and delivery to cytosolic and nuclear client proteins via the cytosolic iron-sulfur cluster assembly (CIA) pathway is not well understood. Here we report cryo-EM structures of the HEAT-repeat protein Met18 from
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1073/pnas.2121491119
