Fe-S Cluster Biogenesis Pathways Research Articles

Structural and Biochemical Characterization of Staphylococcus aureus Cysteine Desulfurase Complex SufSU.

In this work, we provide the first in vitro characterization of two essential proteins from Staphylococcus aureus (S. aureus) involved in iron-sulfur (Fe-S) cluster biogenesis: the cysteine desulfurase SufS and the sulfurtransferase SufU. Together, these proteins form the transient SufSU complex and execute the first stage of Fe-S cluster biogenesis in the SUF-like pathway in Gram-positive bacteria. The proteins involved in the SUF-like pathway, such as SufS and SufU, are essential in Gram-positive bacteria since these bacteria tend to lack redundant Fe-S cluster biogenesis pathways. Most previous work characterizing the SUF-like pathway has focused on Bacillus subtilis (B. subtilis). We focus on the SUF-like pathway in S. aureus because of its potential to serve as a therapeutic target to treat S. aureus infections. Herein, we characterize S. aureus SufS (SaSufS) by X-ray crystallography and UV-vis spectroscopy, and we characterize S. aureus SufU (SaSufU) by a zinc binding fluorescence assay and small-angle X-ray scattering. We show that SaSufS is a type II cysteine desulfurase and that SaSufU is a Zn2+-containing sulfurtransferase. Additionally, we evaluated the cysteine desulfurase activity of the SaSufSU complex and compared its activity to that of B. subtilis SufSU. Subsequent cross-species activity analysis reveals a surprising result: SaSufS is significantly less stimulated by SufU than BsSufS. Our results set a basis for further characterization of SaSufSU as well as the development of new therapeutic strategies for treating infections caused by S. aureus by inhibiting the SUF-like pathway.

Acute loss of iron–sulfur clusters results in metabolic reprogramming and generation of lipid droplets in mammalian cells

Iron-sulfur (Fe-S) clusters are ancient cofactors in cells and participate in diverse biochemical functions, including electron transfer and enzymatic catalysis. Although cell lines derived from individuals carrying mutations in the Fe-S cluster biogenesis pathway or siRNA-mediated knockdown of the Fe-S assembly components provide excellent models for investigating Fe-S cluster formation in mammalian cells, these experimental strategies focus on the consequences of prolonged impairment of Fe-S assembly. Here, we constructed and expressed dominant-negative variants of the primary Fe-S biogenesis scaffold protein iron-sulfur cluster assembly enzyme 2 (ISCU2) in human HEK293 cells. This approach enabled us to study the early metabolic reprogramming associated with loss of Fe-S-containing proteins in several major cellular compartments. Using multiple metabolomics platforms, we observed a ∼12-fold increase in intracellular citrate content in Fe-S-deficient cells, a surge that was due to loss of aconitase activity. The excess citrate was generated from glucose-derived acetyl-CoA, and global analysis of cellular lipids revealed that fatty acid biosynthesis increased markedly relative to cellular proliferation rates in Fe-S-deficient cells. We also observed intracellular lipid droplet accumulation in both acutely Fe-S-deficient cells and iron-starved cells. We conclude that deficient Fe-S biogenesis and acute iron deficiency rapidly increase cellular citrate concentrations, leading to fatty acid synthesis and cytosolic lipid droplet formation. Our findings uncover a potential cause of cellular steatosis in nonadipose tissues.

Abstract B08: Metabolic adaption in inflammatory macrophages through the modulation of Fe-S cluster biogenesis factors

Abstract Macrophages are among the most abundant normal cells in the tumor microenvironment, and the mechanistic overlap in the metabolic changes in glycolytic cancer cells and inflammatory immune cells suggest that insights into the mechanisms underlying the metabolic changes could be useful in the treatment of both cancers and inflammatory diseases. Metabolic choices in immune cells are tightly linked to cell fate and function. Inflammatory immune cells, such as M1 macrophages and T-helper 17 cells, undergo a shift from OXPHOS to enhanced glucose uptake, glycolysis and the pentose phosphate pathway, whereas anti-inflammatory cells, such as M2 macrophages and regulatory T cells have lower glycolytic rates and higher levels of oxidative metabolism. The metabolic switch from OXPHOS to aerobic glycolysis (Warburg effect) in Toll-like receptor (TLR)-stimulated myeloid cells has been shown to result from the activation of glycolysis through the action of AKT and HIF-1α signaling, with mitochondrial respiration passively decreasing due to NF-kB mediated induction of inducible nitric oxide synthase (iNOS), which produces the toxic gas nitric oxide that damages the Fe-S cluster cofactors that are essential for mitochondrial electron transport. The results reported here indicate that the pro-inflammatory activation of macrophages also involved the active suppression of mitochondrial pyruvate catabolism and respiration through the down-regulation of several genes in the Fe-S cluster biogenesis pathway. A decrease in Fe-S cluster biogenesis/repair not only resulted in a decrease in the activities of Fe-S cluster dependent enzymes including the respiratory complexes I and II, mitochondrial and cytosolic aconitases, and ferrochelatase, but also reduced lipoylation in the E2 subunit of pyruvate dehydrogenase (PDH) complex, thereby inhibiting pyruvate catabolism through the TCA cycle. Inhibition of glycolytic metabolism limited the TLR-stimulated repression of Fe-S cluster biogenesis factors, consistent with a vital survival function of Fe-S cluster biogenesis in cells that depend on mitochondrial ATP production. Activation of AMPK, inhibition of mTOR, and inhibition of Nf-kB all resulted in reduced TLR-induced suppression of Fe-S cluster biogenesis factors. These results suggested that the regulation of Fe-S cluster biogenesis factors are highly sensitive to cellular metabolic needs and are subject to the regulation of metabolic regulators AMPK and mTOR and inflammation modulator Nf-kB. Our results suggested that, in addition to iNOS induction, repression of Fe-S cluster biogenesis/repair may serve to suppress OXPHOS to promote the production of mitochondrial ROS for host defense, and to drive prolonged glycolytic metabolism to produce ATP, NADPH, and biosynthetic precursors to meet the metabolic and anabolic demands of host defense. Citation Format: Wing-Hang Tong, Nunziata Maio, Tracey A. Rouault. Metabolic adaption in inflammatory macrophages through the modulation of Fe-S cluster biogenesis factors. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr B08.

The iron-binding CyaY and IscX proteins assist the ISC-catalyzed Fe-S biogenesis in Escherichia coli.

In eukaryotes, frataxin deficiency (FXN) causes severe phenotypes including loss of iron-sulfur (Fe-S) cluster protein activity, accumulation of mitochondrial iron and leads to the neurodegenerative disease Friedreich's ataxia. In contrast, in prokaryotes, deficiency in the FXN homolog, CyaY, was reported not to cause any significant phenotype, questioning both its importance and its actual contribution to Fe-S cluster biogenesis. Because FXN is conserved between eukaryotes and prokaryotes, this surprising discrepancy prompted us to reinvestigate the role of CyaY in Escherichia coli. We report that CyaY (i) potentiates E. coli fitness, (ii) belongs to the ISC pathway catalyzing the maturation of Fe-S cluster-containing proteins and (iii) requires iron-rich conditions for its contribution to be significant. A genetic interaction was discovered between cyaY and iscX, the last gene of the isc operon. Deletion of both genes showed an additive effect on Fe-S cluster protein maturation, which led, among others, to increased resistance to aminoglycosides and increased sensitivity to lambda phage infection. Together, these in vivo results establish the importance of CyaY as a member of the ISC-mediated Fe-S cluster biogenesis pathway in E. coli, like it does in eukaryotes, and validate IscX as a new bona fide Fe-S cluster biogenesis factor.

Coordinate regulation of the Suf and Isc Fe-S cluster biogenesis pathways by IscR is essential for viability of Escherichia coli.

Fe-S cluster biogenesis is essential for the viability of most organisms. In Escherichia coli, this process requires either the housekeeping Isc or the stress-induced Suf pathway. The global regulator IscR coordinates cluster synthesis by repressing transcription of the isc operon by [2Fe-2S]-IscR and activating expression of the suf operon. We show that either [2Fe-2S]-IscR or apo-IscR can activate suf, making expression sensitive to mainly IscR levels and not the cluster state, unlike isc expression. We also demonstrate that in the absence of isc, IscR-dependent suf activation is essential since strains lacking both the Isc pathway and IscR were not viable unless Suf was expressed ectopically. Similarly, removal of the IscR binding site in the sufA promoter also led to a requirement for isc. Furthermore, suf expression was increased in a Δisc mutant, presumably due to increased IscR levels in this mutant. This was surprising because the iron-dependent repressor Fur, whose higher-affinity binding at the sufA promoter should occlude IscR binding, showed only partial repression. In addition, Fur derepression was not sufficient for viability in the absence of IscR and the Isc pathway, highlighting the importance of direct IscR activation. Finally, a mutant lacking Fur and the Isc pathway increased suf expression to the highest observed levels and nearly restored [2Fe-2S]-IscR activity, providing a mechanism for regulating IscR activity under stress conditions. Together, these findings have enhanced our understanding of the homeostatic mechanism by which cells use one regulator, IscR, to differentially control Fe-S cluster biogenesis pathways to ensure viability.

Parasites FeS Up: Iron-Sulfur Cluster Biogenesis in Eukaryotic Pathogens

Iron-sulfur (FeS) clusters are among the most ancient andversatile protein cofactors. They are used by a large and diversegroup of proteins, serving both structural and catalytic roles. Theyfunction in central metabolic processes such as electron transfer,redox chemistry, enzyme catalysis, and sensing environmental orintracellular conditions to regulate gene expression [1,2]. Ferrousiron and sulfur were readily available in the reducing atmospherein which life first evolved, but when oxygen levels rose with theadvent of photosynthetic algae, these building blocks becamescarce. Furthermore, the reactive oxygen species generated asbyproducts of aerobic respiration are highly damaging to FeSclusters, and free iron and sulfide released by FeS clusters are, inturn, toxic to cells. For these reasons, complex mechanismsevolved to coordinate and regulate the biogenesis of these simplecofactors, and these pathways are compartmentalized in theendosymbiotic organelles of eukaryotic cells.All FeS cluster biogenesis pathways follow the same basicprinciples (reviewed in [2]). The first step is the liberation of sulfurby a cysteine desulfurase, which forms a persulfide intermediate ona conserved cysteine residue. Iron is donated by iron-bindingproteins, such as frataxin. The cluster is then assembled on scaffoldproteins with the help of electron donors, which are needed for thereduction of sulfur to sulfide. The fully formed cluster istransferred to apoproteins via chaperones that facilitate the correctsubstrate specificity and proper assembly of the cluster to form themature holoprotein.FeS cluster biogenesis pathways are extremely well conserved,and are invariably essential for viability. Among eukaryoticpathogens, all endosymbiotic organelles studied to date appearto contain FeS cluster biogenesis machinery, and, in some cases,this seems to be the sole reason for retention of the organelle.Typically, the ISC (Iron-Sulfur Cluster formation) pathway residesin the mitochondrion, the CIA (Cytosolic Iron-sulfur proteinAssembly) pathway functions in the cytosol and nucleus, andplastid-containing organisms have an additional pathway, the SUF(SUlFur mobilization) system (Figure 1). In this article, we willcompare the subcellular organization of FeS cluster biogenesispathways in the diverse organelles of eukaryotic pathogens.

Regulation of iron–sulphur cluster homeostasis through transcriptional control of the Isc pathway by [2Fe–2S]–IscR in Escherichia coli

Fe-S clusters are essential across the biological world, yet how cells regulate expression of Fe-S cluster biogenesis pathways to cope with changes in Fe-S cluster demand is not well understood. Here, we describe the mechanism by which IscR, a [2Fe-2S] cluster-containing regulator of Escherichia coli, adjusts the synthesis of the Isc Fe-S biogenesis pathway to maintain Fe-S homeostasis. Our data indicate that a negative feedback loop operates to repress transcription of the iscRSUA-hscBA-fdx operon, encoding IscR and the Isc machinery, through binding of [2Fe-2S]-IscR to two upstream binding sites. IscR was shown to require primarily the Isc pathway for synthesis of its Fe-S cluster, providing a link between IscR activity and demands for Fe-S clusters through the levels of the Isc system. Surprisingly, the isc operon was more repressed under anaerobic conditions, indicating increased Fe-S cluster occupancy of IscR and decreased Fe-S cluster biogenesis demand relative to aerobic conditions. Consistent with this notion, overexpression of a Fe-S protein under aerobic conditions, but not under anaerobic conditions, led to derepression of P(iscR). Together, these data show how transcriptional control of iscRSUA-hscBA-fdx by [2Fe-2S]-IscR allows E. coli to respond efficiently to varying Fe-S demands.

Activation of the Iron Regulon by the Yeast Aft1/Aft2 Transcription Factors Depends on Mitochondrial but Not Cytosolic Iron-Sulfur Protein Biogenesis

Two transcriptional activators, Aft1 and Aft2, regulate iron homeostasis in Saccharomyces cerevisiae. These factors induce the expression of iron regulon genes in iron-deficient yeast but are inactivated in iron-replete cells. Iron inhibition of Aft1/Aft2 is abrogated in cells defective for Fe-S cluster biogenesis within the mitochondrial matrix (Chen, O. S., Crisp, R. J., Valachovic, M., Bard, M., Winge, D. R., and Kaplan, J. (2004) J. Biol. Chem. 279, 29513-29518). To determine whether iron sensing by Aft1/Aft2 requires the function of the mitochondrial Fe-S export and cytosolic Fe-S protein assembly systems, we evaluated the expression of the iron regulon in cells depleted of glutathione and in cells depleted of Atm1, Nar1, Cfd1, and Nbp35. The iron regulon is induced in cells depleted of Atm1 with Aft1 largely responsible for the induced gene expression. Aft2 is activated at a later time in Atm1-depleted cells. Likewise, the iron regulon is induced in cells depleted of glutathione. In contrast, repression of NAR1, CFD1, or NBP35 fails to induce the iron regulon despite strong inhibition of cytosolic/nuclear Fe-S protein assembly. Thus, iron sensing by Aft1/Aft2 is not linked to the maturation of cytosolic/nuclear Fe-S proteins, but the mitochondrial inner membrane transporter Atm1 is important to transport the inhibitory signal. Although Aft1 and Aft2 sense a signal emanating from the Fe-S cluster biogenesis pathway, there is no indication that the proteins are inhibited by direct binding of an Fe-S cluster.

Iron-Sulfur Cluster Biosynthesis

Important for the understanding of the functional properties of the iron-sulfur scaffold IscU is knowledge of the structure and dynamics of this protein class. Structural characterization of Thermotoga maritima IscU by CD (Mansy, S. S., Wu, G., Surerus, K. K., and Cowan, J. A. (2002) J. Biol. Chem. 277, 21397-21404) and high resolution NMR (Bertini, I., Cowan, J. A., Del Bianco, C., Luchinat, C., and Mansy, S. S. (2003) J. Mol. Biol. 331, 907-924) yielded data indicating a high degree of secondary structure. However, the latter also revealed IscU to exist in a dynamic equilibrium between two or more distinct conformations, possibly existing in a molten globule state. Herein, we further characterize the molten globule characteristics of T. maritima IscU by near-ultraviolet circular dichroism, 1-anilino-8-naphthalenesulfonic acid binding, free energy of unfolding, hydrodynamic radius measurements, and limited tryptic digestion. The data suggest unusual dynamic behavior that is not fully consistent with typical protein states such as fully folded, fully unfolded, or molten globule. For instance, the existence of a stable tertiary fold is supported by near-UV CD spectra and hydrodynamic radius measurements, whereas other data are less clearly interpretable and may be viewed as consistent with either a molten globule or fully folded state. However, all of the data are consistent with our previous hypothesis of a protein sampling multiple discrete tertiary conformations in which these structural transitions occur on a "slow" time scale. To describe such proteins, we introduce the term multiple discrete conformers.