Myeloperoxidase-derived oxidants inhibit sarco/endoplasmic reticulum Ca2+-ATPase activity and perturb Ca2+ homeostasis in human coronary artery endothelial cells

The sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) plays a critical role in Ca(2+) homeostasis via sequestration of this ion in the sarco/endoplasmic reticulum. The activity of this pump is inhibited by oxidants and impaired in aging tissues and cardiovascular disease. We have shown previously that the myeloperoxidase (MPO)-derived oxidants HOCl and HOSCN target thiols and mediate cellular dysfunction. As SERCA contains Cys residues critical to ATPase activity, we hypothesized that HOCl and HOSCN might inhibit SERCA activity, via thiol oxidation, and increase cytosolic Ca(2+) levels in human coronary artery endothelial cells (HCAEC). Exposure of sarcoplasmic reticulum vesicles to preformed or enzymatically generated HOCl and HOSCN resulted in a concentration-dependent decrease in ATPase activity; this was also inhibited by the SERCA inhibitor thapsigargin. Decomposed HOSCN and incomplete MPO enzyme systems did not decrease activity. Loss of ATPase activity occurred concurrent with oxidation of SERCA Cys residues and protein modification. Exposure of HCAEC, with or without external Ca(2+), to HOSCN or HOCl resulted in a time- and concentration-dependent increase in intracellular Ca(2+) under conditions that did not result in immediate loss of cell viability. Thapsigargin, but not inhibitors of plasma membrane or mitochondrial Ca(2+) pumps/channels, completely attenuated the increase in intracellular Ca(2+) consistent with a critical role for SERCA in maintaining endothelial cell Ca(2+) homeostasis. Angiotensin II pretreatment potentiated the effect of HOSCN at low concentrations. MPO-mediated modulation of intracellular Ca(2+) levels may exacerbate endothelial dysfunction, a key early event in atherosclerosis, and be more marked in smokers because of their higher SCN(-) levels.     

Recycling of RNA binding iron regulatory protein 1 into an aconitase after nitric oxide removal depends on mitochondrial ATP

Iron regulatory proteins (IRPs) control iron metabolism by specifically interacting with iron-responsive elements (IREs) on mRNAs. Nitric oxide (NO) converts IRP-1 from a [4Fe-4S] aconitase to a trans-regulatory protein through Fe-S cluster disassembly. Here, we have focused on the fate of IRE binding IRP1 from murine macrophages when NO flux stops. We show that virtually all IRP-1 molecules from NO-producing cells dissociated from IRE and recovered aconitase activity after re-assembling a [4Fe-4S] cluster in vitro. The reverse change in IRP-1 activities also occurred in intact cells no longer exposed to NO and did not require de novo protein synthesis. Likewise, inhibition of mitochondrial aconitase via NO-induced Fe-S cluster disassembly was also reversed independently of protein translation after NO removal. Our results provide the first evidence of Fe-S cluster repair of NO-modified aconitases in mammalian cells. Moreover, we show that reverse change in IRP-1 activities and repair of mitochondrial aconitase activity depended on energized mitochondria. Finally, we demonstrate that IRP-1 activation by NO was accompanied by both a drastic decrease in ferritin levels and an increase in transferrin receptor mRNA levels. However, although ferritin expression was recovered upon IRP-1-IRE dissociation, expression of transferrin receptor mRNA continued to rise for several hours after stopping NO flux.     

Modulation of iron regulatory protein-1 by various metals.

Iron regulatory protein-1 (IRP-1) is known as a cytosolic aconitase and a central regulator of iron (Fe) homeostasis. IRP-1 regulates the expression of Fe metabolism-related proteins by interacting with the Fe-responsive element (IRE) in the untranslated regions of mRNAs of these proteins. However, it is less known whether IRP-1 modulates various non-Fe metals. In the present study, we showed that treatment of homogenously purified IRP-1 with non-Fe metals decreased the affinity to IRE in RNA band shift assays and increased aconitase activity. Non-Fe metals also inhibited (55)Fe incorporation into the fourth labile position of the Fe-S cluster of IRP-1. In PLC hepatoma cells, metal loading inactivated binding activity and activated enzyme activity. It also suppressed transferrin receptor mRNA expression in the cells. These results suggest that various non-Fe metals modulate IRP-1 by conversion of the 3Fe-4S apo-form to a [1 non-Fe metal + 3Fe]-4Fe holo-form.     

Inactivation of both RNA binding and aconitase activities of iron regulatory protein-1 by quinone-induced oxidative stress

Iron regulatory protein-1 (IRP-1) controls the expression of several mRNAs by binding to iron-responsive elements (IREs) in their untranslated regions. In iron-replete cells, a 4Fe-4S cluster converts IRP-1 to cytoplasmic aconitase. IRE binding activity is restored by cluster loss in response to iron starvation, NO, or extracellular H2O2. Here, we study the effects of intracellular quinone-induced oxidative stress on IRP-1. Treatment of murine B6 fibroblasts with menadione sodium bisulfite (MSB), a redox cycling drug, causes a modest activation of IRP-1 to bind to IREs within 15-30 min. However, IRE binding drops to basal levels within 60 min. Surprisingly, a remarkable loss of both IRE binding and aconitase activities of IRP-1 follows treatment with MSB for 1-2 h. These effects do not result from alterations in IRP-1 half-life, can be antagonized by the antioxidant N-acetylcysteine, and regulate IRE-containing mRNAs; the capacity of iron-starved MSB-treated cells to increase transferrin receptor mRNA levels is inhibited, and MSB increases the translation of a human growth hormone indicator mRNA bearing an IRE in its 5'-untranslated region. Nonetheless, MSB inhibits ferritin synthesis. Thus, menadione-induced oxidative stress leads to post-translational inactivation of both genetic and enzymatic functions of IRP-1 by a mechanism that lies beyond the "classical" Fe-S cluster switch and exerts multiple effects on cellular iron metabolism.     

Cytosolic aconitase and ferritin are regulated by iron in Caenorhabditis elegans

Iron regulatory protein-1 (IRP-1) is a cytosolic RNA-binding protein that is a regulator of iron homeostasis in mammalian cells. IRP-1 binds to RNA structures, known as iron-responsive elements, located in the untranslated regions of specific mRNAs, and it regulates the translation or stability of these mRNAs. Iron regulates IRP-1 activity by converting it from an RNA-binding apoprotein into a [4Fe-4S] cluster protein exhibiting aconitase activity. IRP-1 is widely found in prokaryotes and eukaryotes. Here, we report the biochemical characterization and regulation of an IRP-1 homolog in Caenorhabditis elegans (GEI-22/ACO-1). GEI-22/ACO-1 is expressed in the cytosol of cells of the hypodermis and the intestine. Like mammalian IRP-1/aconitases, GEI-22/ACO-1 exhibits aconitase activity and is post-translationally regulated by iron. Although GEI-22/ACO-1 shares striking resemblance to mammalian IRP-1, it fails to bind RNA. This is consistent with the lack of iron-responsive elements in the C. elegans ferritin genes, ftn-1 and ftn-2. While mammalian ferritin H and L mRNAs are translationally regulated by iron, the amounts of C. elegans ftn-1 and ftn-2 mRNAs are increased by iron and decreased by iron chelation. Excess iron did not significantly alter worm development but did shorten their life span. These studies indicated that iron homeostasis in C. elegans shares some similarities with those of vertebrates.     

High plasma thiocyanate levels are associated with enhanced myeloperoxidase-induced thiol oxidation and long-term survival in subjects following a first myocardial infarction

Abstract

Elevated levels of myeloperoxidase (MPO) are associated with poor cardiovascular outcomes. MPO uses H₂O₂ to generate oxidants including HOCl and HOSCN, from chloride and thiocyanate (SCN^?) ions, respectively. SCN^? is the preferred substrate. Elevation of this anion decreases HOCl generation and increases HOSCN formation, a thiol-specific oxidant. Such changes are of potential relevance to people with elevated SCN^? levels, such as smokers. In this retrospective study, we examined whether elevated plasma MPO and SCN^? levels increased thiol oxidation as a result of increased HOSCN formation, and impacted on long-term survival in 176 subjects (74 non-smokers, 46 smokers, and 56 previous smokers) hospitalized after a first myocardial infarction. Plasma thiols were not significantly altered in smokers compared to non-smokers or past smokers. However, significant positive correlations were detected between SCN^? levels and MPO-induced thiol loss in the total population (r = 0.19, P = 0.020) and smokers alone (r = 0.58, P < 0.0001). Twelve-year all-cause mortality data indicate that above median MPO is significantly associated with higher mortality, but below-median MPO and above-median SCN^? results in increased survival, compared to below-median SCN^?. Cox proportional hazard analysis showed a significant decrease in mortality for each 1 μM increase in SCN^? (0.991; P = 0.040). Subject age was, as expected, a strong predictor of subject survival. Overall these data suggest that subjects with below-median MPO and above-median SCN^? have better long-term survival, and that elevated plasma levels of SCN^? might be protective in at least some populations.     

Ligand-induced structural alterations in human iron regulatory protein-1 revealed by protein footprinting.

Human iron regulatory protein-1 (IRP-1) is a bifunctional protein that regulates iron metabolism by binding to mRNAs encoding proteins involved in iron uptake, storage, and utilization. Intracellular iron accumulation regulates IRP-1 function by promoting the assembly of an iron-sulfur cluster, conferring aconitase activity to IRP-1, and hindering RNA binding. Using protein footprinting, we have studied the structure of the two functional forms of IRP-1 and have mapped the surface of the iron-responsive element (IRE) binding site. Binding of the ferritin IRE or of the minimal regulatory region of transferrin receptor mRNA induced strong protections against proteolysis in the region spanning amino acids 80 to 187, which are located in the putative cleft thought to be involved in RNA binding. In addition, IRE-induced protections were also found in the C-terminal domain at Arg-721 and Arg-728. These data implicate a bipartite IRE binding site located in the putative cleft of IRP-1. The aconitase form of IRP-1 adopts a more compact structure because strong reductions of cleavage were detected in two defined areas encompassing residues 149 to 187 and 721 to 735. Thus both ligands of apo-IRP-1, the IRE and the 4Fe-4S cluster, induce distinct but overlapping alterations in protease accessibility. These data provide evidences for structural changes in IRP-1 upon cluster formation that affect the accessibility of residues constituting the RNA binding site.     

Global Identification of Genes Affecting Iron-Sulfur Cluster Biogenesis and Iron Homeostasis

Iron-sulfur (Fe-S) clusters are ubiquitous cofactors that are crucial for many physiological processes in all organisms. In Escherichia coli, assembly of Fe-S clusters depends on the activity of the iron-sulfur cluster (ISC) assembly and sulfur mobilization (SUF) apparatus. However, the underlying molecular mechanisms and the mechanisms that control Fe-S cluster biogenesis and iron homeostasis are still poorly defined. In this study, we performed a global screen to identify the factors affecting Fe-S cluster biogenesis and iron homeostasis using the Keio collection, which is a library of 3,815 single-gene E. coli knockout mutants. The approach was based on radiolabeling of the cells with [2-¹⁴C]dihydrouracil, which entirely depends on the activity of an Fe-S enzyme, dihydropyrimidine dehydrogenase. We identified 49 genes affecting Fe-S cluster biogenesis and/or iron homeostasis, including 23 genes important only under microaerobic/anaerobic conditions. This study defines key proteins associated with Fe-S cluster biogenesis and iron homeostasis, which will aid further understanding of the cellular mechanisms that coordinate the processes. In addition, we applied the [2-¹⁴C]dihydrouracil-labeling method to analyze the role of amino acid residues of an Fe-S cluster assembly scaffold (IscU) as a model of the Fe-S cluster assembly apparatus. The analysis showed that Cys37, Cys63, His105, and Cys106 are essential for the function of IscU in vivo, demonstrating the potential of the method to investigate in vivo function of proteins involved in Fe-S cluster assembly.     

Functions of mitochondrial ISCU and cytosolic ISCU in mammalian iron-sulfur cluster biogenesis and iron homeostasis

Iron-sulfur (Fe-S) clusters are required for the functions of mitochondrial aconitase, mammalian iron regulatory protein 1, and many other proteins in multiple subcellular compartments. Recent studies in Saccharomyces cerevisiae indicated that Fe-S cluster biogenesis also has an important role in mitochondrial iron homeostasis. Here we report the functional analysis of the mitochondrial and cytosolic isoforms of the human Fe-S cluster scaffold protein, ISCU. Suppression of human ISCU by RNAi not only inactivated mitochondrial and cytosolic aconitases in a compartment-specific manner but also inappropriately activated the iron regulatory proteins and disrupted intracellular iron homeostasis. Furthermore, endogenous ISCU levels were suppressed by iron deprivation. These results provide evidence for a coordinated response to iron deficiency that includes activation of iron uptake, redistribution of intracellular iron, and decreased utilization of iron in Fe-S proteins.     

Structural investigations by extended X-ray absorption fine structure spectroscopy of the iron center of mitochondrial aconitase in higher plant cells.

We have obtained iron K-edge extended x-ray absorption fine structure spectra of the plant mitochondrial aconitase in its active state, in the presence (aconitase (+)) and absence (aconitase (-)) of the substrate citrate. Analysis of the data indicates that oxygens are present in the first coordination shell, at an average Fe-O distance of 1.96/1.98 A (aconitase (+)/aconitase(-)). Part of these oxygens is provided by the citrate, which binds at 1.99 A from the iron in aconitase (+). The second shell (sulfur) contribution is split and is consistent with Fe-S distances of 2.30/2.29 and 2.56/2.59 A, and the third shell (iron) is consistent with an Fe-Fe distance of 2.83/2.84 A. Both Fe-S and Fe-Fe distances are longer than similar distances found in most Fe-S centers. A strong scattering at approximately 5 A has been identified as originating from an iron atom which is near to, but not part of, the Fe-S cluster. These data indicate that active plant mitochondrial aconitase contains a novel type of iron center.     

Sites and mechanisms of aconitase inactivation by peroxynitrite: modulation by citrate and glutathione

Aconitases are iron-sulfur cluster-containing proteins present both in mitochondria and cytosol of cells; the cubane iron-sulfur (Fe-S) cluster in the active site is essential for catalytic activity, but it also renders aconitase highly vulnerable to reactive oxygen and nitrogen species. This study examined the sites and mechanisms of aconitase inactivation by peroxynitrite (ONOO-), a strong oxidant and nitrating agent readily formed from superoxide anion and nitric oxide generated by mitochondria. ONOO- inactivated aconitase in a dose-dependent manner (half-maximal inhibition was observed with approximately 3 microM ONOO-). Low levels of ONOO- caused the conversion of the Fe-S cluster from the [4Fe-4S]2+ form to the inactive [3Fe-4S]1+ form with the loss of labile iron, as confirmed by low-temperature EPR analysis. In the presence of the substrate, citrate, 66-fold higher concentrations of ONOO- were required for half-maximal inhibition. The protective effects of citrate corresponded to its binding to the active site. The inactivation of aconitase in the presence of citrate was due to ONOO--mediated cysteine thiol loss and tyrosine nitration in the enzyme as shown by Western blot analyses. LC/MS/MS analyses revealed that ONOO- treatment to aconitase resulted in nitration of tyrosines 151 and 472 and oxidation to sulfonic acid of cysteines 126 and 385. The latter is one of the three cysteine residues in aconitase that binds to the Fe-S cluster. All other modified tyrosine and cysteine residues were adjacent to the binding site, thus suggesting that these modifications caused conformational changes leading to active-site disruption. Aconitase cysteine thiol modifications other than oxidation to sulfonic acid, such as S-glutathionylation, also decreased aconitase activity, thus indicating that glutathionylation may be an important means of modulating aconitase activity under oxidative and nitrative stress. Taken together, these results demonstrate that the Fe-S cluster in the active site, cysteine 385 bound to the Fe-S cluster, and tyrosine and cysteine residues in the vicinity of the active site are important targets of oxidative and/or nitrative attack, which is selectively controlled by the mitochondrial matrix citrate levels. The mechanisms inherent in aconitase inactivation by ONOO- are discussed in terms of the mitochondrial matrix metabolic and thiol redox state.     

Superoxide-Driven Aconitase FE–S Center Cycling

O^- ₂ produced by the autoxidation of respiratory chain electron carriers, and other cellular reductants, inactivates bacterial and mammalian iron-sulfur-containing (de)hydratases including the citric acid cycle enzyme aconitase. Release of the solvent-exposed iron atom and oxidation of the [4Fe-4S]²⁺ cluster accompanies loss of catalytic activity. Rapid reactivation is achieved by iron-sulfur cluster reduction and Fe²⁺ insertion. Inactivation-reactivation is a dynamic and cyclical process which modulates aconitase and (de)hydratase activities in Escherichia coli and mammalian cells. The balance of inactive and active aconitase provides a sensitive measure of the changes in steady-statO^- ₂ levels occuring in living cells and mitochondria under stress conditions. Aconitases are also inactivated by other oxidants including O₂, H₂O₂, NO^., and ONOO^– which are associated with inflammation, hyperoxia and other pathophysiological conditions. Loss of aconitase activity during oxidant stress may impair energy production, and the liberation of reactive iron may further enhance oxidative damage. Iron-sulfur center cycling may also serve adaptive functions by modulating gene expression or by signaling metabolic quiescence.     

Metabolic control analysis of mitochondrial aconitase: influence over respiration and mitochondrial superoxide and hydrogen peroxide production

Abstract

The Fe-S cluster of mitochondrial aconitase is rapidly and selectively inactivated by oxidants, yielding an inactive enzyme that can be reactivated by reductants and iron in vivo. In order to elucidate the metabolic impact of oxidant-dependent aconitase inhibition over the citric acid cycle, the respiratory chain reactions, and reactive species formation, we performed a metabolic analysis using isolated mitochondria from different rat tissues. Titrations with fluorocitrate showed IC₅₀ for aconitase inhibition ranging from 7 to 24 μM. The aconitase inhibition threshold in mitochondrial oxygen consumption was determined to range from 63 to 98%. Of the tissues examined, brain and heart exhibited the highest values in the flux control coefficient (> 0.95). Aconitase-specific activity varied widely among tissues examined from ?60 mU/mg in liver to 321 mU/mg in kidney at 21% O₂. In brain and heart, aconitase-specific activity increased by 42 and 12%, respectively, at 2% O₂ reflecting aconitase inactivation by oxygen-derived oxidants at 21% O₂. Both mitochondrial membrane potential and hydrogen peroxide production significantly decreased upon aconitase inhibition in heart and brain mitochondria. These results indicate that aconitase can exert control over respiration (with tissue specificity) and support the hypothesis that inactivation of aconitase may provide a control mechanism to prevent O₂^●? and H₂O₂ formation by the respiratory chain.