Transcriptional regulation of FeS biogenesis genes: A possible shield against arsenate toxicity activated by Yap1.

In the eukaryotic model yeast Saccharomyces cerevisiae, arsenic (As) detoxification is regulated by two transcriptional factors, Yap8 and Yap1. Yap8 specifically controls As extrusion from the cell, whether Yap1 avoids arsenic-induced oxidative damages. Accordingly, cells lacking both Yap1 and Yap8 are more sensitive to arsenate than cells lacking each regulator individually. Strikingly enough, the same sensitivity pattern was observed under anoxia, suggesting that Yap1 role in As detoxification might not be restricted to the regulation of the oxidative stress response. This finding prompted us to study the transcriptomic profile of wild-type and yap1 mutant cells exposed to arsenate. Interestingly, we found that, under such conditions, several genes involved in the biogenesis of FeS proteins were upregulated in a Yap1-dependent way. In line with this observation, arsenate treatment decreases the activity of the mitochondrial aconitase, Aco1, an FeS cluster-containing enzyme, this effect being even more pronounced in the yap1 mutant. Reinforcing the relevance of FeS cluster biogenesis in arsenate detoxification, the overexpression of several ISC and CIA machinery genes alleviates the deleterious effect of arsenate caused by the absence of Yap1 and Yap8. Altogether our data suggest that the upregulation of FeS biogenesis genes regulated by Yap1 might work as a cellular shield against arsenate toxicity.

Comparison of the Response of Bacterial IscU and SufU to Zn2+ and Select Transition-Metal Ions.

IscU, the central scaffold protein in the bacterial ISC iron-sulfur (Fe-S) cluster biosynthesis system, has long been recognized to bind a Zn2+ ion at its active site. While initially regarded as an artifact, Zn2+ binding has been shown to induce stabilization of the IscU structure that may mimic a state biologically relevant to IscU's role in Fe-S cluster biosynthesis. More recent studies have revealed that SufU, a homologous protein involved in Fe-S cluster biosynthesis in Gram-positive bacteria, also binds a Zn2+ ion with structural implications. Given the widespread occurrence of the "IscU-like" protein fold, particularly among Fe-S cluster biosynthesis systems, an interesting question arises as to whether Zn2+ ion binding and the resulting structural alterations are common properties in IscU-like proteins. Interactions between IscU and specific metal ions are investigated and compared side-by-side with those of SufU from a representative Gram-positive bacterium in the phylum Firmicutes. These studies were extended with additional transition metal ions chosen to investigate the influence of coordination geometry on selectivity for binding at the active sites of IscU and SufU. Monitoring and comparing the conformational behavior and stabilization afforded by different transition metal ions upon IscU and SufU revealed similarities between the two proteins and suggest that metal-dependent conformational transitions may be characteristic of U-type proteins involved in Fe-S cluster biosynthesis.

Novel mutations in ETFDH gene in Chinese patients with riboflavin-responsive multiple acyl-CoA dehydrogenase deficiency.

BACKGROUND: Multiple acyl-CoA dehydrogenase deficiency (MADD, OMIM 231680) or glutaric aciduria type II (GAII) is an inherited autosomal recessive disease affecting fatty acid, amino acid and choline metabolism, due to mutations in one of three genes namely, electron transfer flavoprotein alpha-subunit, ETFA (OMIM 608053), electron transfer flavoprotein beta-subunit, ETFB (OMIM 130410) and electron transfer flavoprotein dehydrogenase, ETFDH (OMIM 231675). Some MADD patients are responsive to riboflavin treatment with an excellent prognosis. Recently, mutations in ETFDH were found to be responsible for all riboflavin-responsive MADD patients. In this study, we present the clinical features and molecular studies of 2 Chinese families with riboflavin-responsive MADD. METHODS: Genomic DNA was extracted from peripheral blood samples or skin fibroblast cultures from the patients and normal controls. The thirteen exons of ETFDH were amplified by PCR. PCR products were sequenced in both forward and reverse directions. To rule out mutations in other genes, phenotype segregation was studied in the families by microsatellite markers in the proximity of the 3 genes, ETFA, ETFB and ETFDH. RESULTS: Four novel mutations in ETFDH were detected in the 2 families. In family 1, a frame shift mutation, c.1355delG which introduced a premature-termination codon (PTC), I454X in exon 11 of ETFDH was found. Another mutation was a c.250G>A transition in exon 3 of ETFDH, A84T. In family 2, two novel missense mutations were identified, P137S, in exon 4 and G467R in exon 11. No carrier of these four mutations was identified from about 150 alleles of healthy Chinese control subjects. CONCLUSIONS: Four novel mutations (3 missenses and 1 deletion) in ETFDH were found in Chinese families that presented with riboflavin-responsive MADD, which further expands the list of mutations found in patients with riboflavin-responsive MADD. Furthermore, we illustrated the utility of phenotype-genotype segregation in MADD families to prioritize genes for sequencing or to rule out the presence of disease causing mutation in other genes in MADD and other diseases caused by multiple genes.

Probing the role of copper in the biosynthesis of the molybdenum cofactor in Escherichia coli and Rhodobacter sphaeroides.

The crystal structure of Cnx1G, an enzyme involved in the biosynthesis of the molybdenum cofactor (Moco) in Arabidopsis thaliana, revealed the remarkable feature of a copper ion bound to the dithiolene unit of a molybdopterin intermediate (Kuper et al. Nature 430:803-806, 2004). To characterize further the role of copper in Moco biosynthesis, we examined the in vivo and/or in vitro activity of two Moco-dependent enzymes, dimethyl sulfoxide reductase (DMSOR) and nitrate reductase (NR), from cells grown under a variety of copper conditions. We found the activities of DMSOR and NR were not affected when copper was depleted from the media of either Escherichia coli or Rhodobacter sphaeroides. These data suggest that while copper may be utilized during Moco biosynthesis when it is available, copper does not appear to be strictly required for Moco biosynthesis in these two organisms.

Fe-S cluster proteins are intracellular targets for nitric oxide generated luminally at the gastro-oesophageal junction.

In human, high concentrations of nitric oxide are generated at the gastro-oesophageal junction through entero-salivary recirculation of dietary nitrate. Nitric oxide is known to have a high affinity for Fe-S cluster proteins. The aim of this study is to investigate whether nitric oxide arising from the lumen diffuses into the adjacent tissue where it can interact with Fe-S proteins both in a rat animal model and human. An electron paramagnetic resonance detectable complex, dinitrosyl dithiolato iron complex (DNIC), was used as a biomarker for the interaction between Fe-S proteins and nitric oxide. The generation of the complex was evaluated in resected gastric tissue of nitrite-administered rat or biopsy specimens from human after nitrate ingestion. The activity of aconitase, one of the Fe-S cluster proteins, was also determined. The signal of the complex was observed at the rat gastro-oesophageal junction where luminal generation of nitric oxide from nitrite was maximal, and the intensity increased in a dose- and time-dependent manner. The appearance of the complex was accompanied by a significant inhibition of the aconitase activity at that site. The complex appeared in biopsy specimens from the gastro-oesophageal junction in three of five men after nitrate ingestion. Since DNIC is considered to be a decomposition product when Fe-S cluster proteins interact with nitric oxide, the appearance of the signal provides direct evidence that nitric oxide arising from the lumen can destroy such proteins. DNIC formation may represent the cellular mechanism responsible for the high prevalence of disease at the gastro-oesophageal junction.

Iron-sulphur clusters and the problem with oxygen.

During the first billion years of life on the Earth, the environment was anaerobic. Iron and sulphur were plentiful, and they were recruited in the formation of iron-sulphur (Fe-S) clusters within ancient proteins. These clusters provided many enzymes with the ability to transfer electrons; to others they offered a cationic feature that tightly bound oxyanionic and nitrogenous metabolites. Still others acquired a crystallizing surface around which polypeptide could fold to establish a three-dimensional structure. However, the subsequent oxygenation of the Earth's atmosphere by photosynthetic organisms created a threat to cluster-dependent proteins that still has not been fully resolved. By oxidizing environmental iron, oxygen limits its bioavailability, requiring that organisms employ complex schemes with which to satisfy their iron requirement. More directly, oxygen species convert exposed Fe-S clusters to unstable forms that quickly decompose. Some microbes responded to this dilemma by retreating to anaerobic habitats. Others abandoned the use of low-potential electron-transfer pathways, which rely upon the least stable cluster enzymes, and developed antioxidant strategies to protect the remainder. These adjustments were only partially successful: largely because of their reliance upon Fe-S clusters, aerobes remain vulnerable to iron restriction and oxidative stress, features that higher organisms exploit in defending themselves against bacterial pathogens. Thus, the history of Fe-S clusters is an unusual one that has profoundly shaped contemporary microbial ecology.

Effect of mexidol and nitroglycerine on iron-sulfur centers, cytochrome P-450, and nitric oxide formation in liver tissue of experimental animals.

In experiments on mice we studied the effect of individual or combined treatment with mexidol and nitroglycerine on iron-sulfur centers of the mitochondrial respiratory chain, cytochrome P-450 of the endoplasmic reticulum, and nitric oxide formation in the liver tissue. Mexidol had a potent effect on these parameters and protected iron-sulfur centers from oxidation, including that induced by nitroglycerine.

Unexpected anthracycline-mediated alterations in iron-regulatory protein-RNA-binding activity: the iron and copper complexes of anthracyclines decrease RNA-binding activity.

Anthracyclines are effective antineoplastic agents. However, the interaction of these drugs with iron (Fe) is an important cause of myocardial toxicity, limiting their therapeutic use (J Lab Clin Med 122:245-251, 1993). To overcome this limitation, it is crucial to understand how anthracyclines interact with the Fe metabolism of myocardial and neoplastic cells. Iron-regulatory proteins (IRPs) play vital roles in regulating cellular Fe metabolism via their mRNA-binding activity. We showed that doxorubicin (DOX) and its analogs interfere with tumor and myocardial cell Fe metabolism by affecting the RNA-binding activity of IRPs. Unexpectedly, experiments with the free radical scavengers, catalase, superoxide dismutase, ebselen, and Mn(III) tetrakis (4-benzoic acid) porphyrin complex, suggested that the effects of DOX on IRP-RNA-binding activity were not due to anthracycline-mediated free radical production. In contrast to previous studies, we showed that the DOX metabolite, doxorubicinol, had no effect on IRP-RNA-binding activity. Rather, the anthracycline-Fe and -copper (Cu) complexes decreased IRP-RNA-binding activity, indicating that formation of anthracycline-metal complexes may affect cellular Fe metabolism. In addition, anthracyclines prevented the response of IRPs to the depletion of intracellular Fe by chelators. This information may be useful in designing novel therapeutic strategies against tumor cells by combining chelators and anthracyclines. Interestingly, the effect of DOX on primary cultures of cardiomyocytes was similar to that observed using neoplastic cells, and particularly notable was the decrease in IRP2-RNA-binding activity. Our results add significant new information regarding the effects of anthracyclines on Fe metabolism that may lead to the design of more effective treatments.

Conditional derepression of ferritin synthesis in cells expressing a constitutive IRP1 mutant.

Iron regulatory protein 1 (IRP1), a major posttranscriptional regulator of cellular iron and energy metabolism, is controlled by an iron-sulfur cluster switch. Cysteine-437 is critical for coordinating the cluster, and its replacement yields mutants that do not respond to iron perturbations and constitutively bind to cognate mRNA iron-responsive elements (IREs). The expression of IRP1(C437S) in cells has been associated with aberrations in iron homeostasis and toxicity. We have established clones of human lung (H1299) and breast (MCF7) cancer cells that express high levels of IRP1(C437S) in a tetracycline-inducible manner. As expected, IRP1(C437S) stabilizes transferrin receptor mRNA and inhibits translation of ferritin mRNA in both cell types by binding to their respective IREs. However, H1299 transfectants grown at high densities are able to overcome the IRP1(C437S)-mediated inhibition in ferritin synthesis. The mechanism involves neither alteration in ferritin mRNA levels nor utilization of alternative transcription start sites to eliminate the IRE or relocate it in less inhibitory downstream positions. The derepression of ferritin mRNA translation occurs under conditions where global protein synthesis appears to be impaired, as judged by a significant enrichment in the expression of the underphosphorylated form of the translational regulator 4E-BP1. Collectively, these data document an example where ferritin mRNA translation evades control of the IRE-IRP system. The physiological implications of this response are reflected in protection against iron-mediated toxicity, oxidative stress, and apoptosis.

Control of FNR function of Escherichia coli by O2 and reducing conditions.

The synthesis of the enzymes constituting the electron transport chain of Escherichia coli is controlled by electron acceptors in order to achieve high ATP yields and high metabolic rates as well. High ATP yields (or efficiency) are obtained by the use of electron acceptors for respiration which allow high ATP yields, preferentially O2, and nitrate in the absence of O2. The rate of metabolism is adjusted by use of respiratory isoenzymes which differ in the rate and the efficiency of energy conservation, such as the non-coupling NADH dehydrogenase II (ndh gene) and the coupling NADH dehydrogenase I (nuo genes). By combination of the contrary principles (rate versus efficiency), growth is optimized for growth yields and rates. One of the major transcriptional regulators controlling the switch from aerobic to anaerobic respiration is FNR (fumarate nitrate reductase regulator). FNR is located in the cytoplasm and contains a [4Fe-4S] cluster in the active (anaerobic) state. By reaction with O2 the cluster is converted to a [2Fe-2S] cluster and finally to apoFNR. O2 diffuses into the cytoplasm even at very low O2-tensions (1 microM) where it inactivates [4Fe-4S] x FNR. The formation of [4Fe-4S] x FNR from apoFNR can use glutathione as a reducing agent in vitro. This process could also be important for the reductive activation of FNR in vivo. A model for the control of the functional state of FNR by O2 and glutathione is discussed. According to this model the functional state of FNR is determined by a (rapid) inactivation of FNR by O2, and a slow (constant) reactivation with glutathione as the reducing agent.

Iron treatment downregulates DMT1 and IREG1 mRNA expression in Caco-2 cells.

Iron deficiency is the most common nutritional disorder worldwide, whereas pathologic elevations of body iron stores can occur under certain circumstances due to genetic abnormalities or in association with other diseases. The intestine is the exclusive locus of homeostatic regulation of body iron stores, which is accomplished by changes in iron absorption efficiency by largely unknown molecular mechanisms in response to alterations in body iron stores. Recently, a number of novel genes involved in iron metabolism, such as the iron uptake transporter DMT1/DCT1/Nramp2 and the iron export transporter IREG1/ferroportin1/MTP1, have been identified, providing important insights about molecular aspects of intestinal iron absorption and its regulation. The aim of this study was to investigate the effects of iron treatment on DMT1 and IREG1 mRNA expression in Caco-2 cells, a human intestinal cell line. Exposure of the cells to iron (200 micromol/L ferric nitrilotriacetic acid for 72 h) significantly decreased transferrin receptor mRNA (80%), DMT1 mRNA (57%) and IREG1 mRNA (52%). These observations are consistent with the notion of parallel regulation of these iron-responsive genes in vivo to protect the enterocyte from iron toxicity and mediate a decreased efficiency of intestinal iron absorption to prevent iron overload.

Biochemical and morphological characterization of sulfur-deprived and H2-producing Chlamydomonas reinhardtii (green alga).

Sulfur deprivation in green algae causes reversible inhibition of photosynthetic activity. In the absence of S, rates of photosynthetic O2 evolution drop below those of O2 consumption by respiration. As a consequence, sealed cultures of the green alga Chlamydomonas reinhardtii become anaerobic in the light, induce the "Fe-hydrogenase" pathway of electron transport and photosynthetically produce H2 gas. In the course of such H2-gas production cells consume substantial amounts of internal starch and protein. Such catabolic reactions may sustain, directly or in directly, the H2-production process. Profile analysis of selected photosynthetic proteins showed a precipitous decline in the amount of ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) as a function of time in S deprivation, a more gradual decline in the level of photosystem (PS) II and PSI proteins, and a change in the composition of the PSII light-harvesting complex (LHC-II). An increase in the level of the enzyme Fe-hydrogenase was noted during the initial stages of S deprivation (0-72 h) followed by a decline in the level of this enzyme during longer (t >72 h) S-deprivation times. Microscopic observations showed distinct morphological changes in C. reinhardtii during S deprivation and H2 production. Ellipsoid-shaped cells (normal photosynthesis) gave way to larger and spherical cell shapes in the initial stages of S deprivation and H2 production, followed by cell mass reductions after longer S-deprivation and H2-production times. It is suggested that, under S-deprivation conditions, electrons derived from a residual PSII H2O-oxidation activity feed into the hydrogenase pathway, thereby contributing to the H2-production process in Chlamydomonas reinhardtii. Interplay between oxygenic photosynthesis, mitochondrial respiration, catabolism of endogenous substrate, and electron transport via the hydrogenase pathway is essential for this light-mediated H2-production process.

N-acetylcysteine inhibits in vivo nitric oxide production by inducible nitric oxide synthase.

This in vivo study evaluates the effect of N-acetylcysteine (NAC) administration on nitric oxide (NO) production by the inducible form of nitric oxide synthase (iNOS). NO production was induced in the rat by the ip administration of 2 mg/100 g lipopolysaccharide (LPS). This treatment caused: (1) a decrease in body temperature within 90 min, followed by a slow return to normal levels; (2) an increase in plasma levels of urea, nitrite/nitrate, and citrulline; (3) the appearance in blood of nitrosyl-hemoglobin (NO-Hb) and in liver of dinitrosyl-iron-dithiolate complexes (DNIC); and (4) increased expression of iNOS mRNA in peripheral blood mononuclear cells (PBMC). Rat treatment with 15 mg/100 g NAC ip, 30 min before LPS, resulted in a significant decrease in blood NO-Hb levels, plasma nitrite/nitrate and citrulline concentrations, and liver DNIC complexes. PBMC also showed a decreased expression of iNOS mRNA. NAC pretreatment did not modify the increased levels of plasma urea or the hypothermic effect induced by the endotoxin. The administration of NAC following LPS intoxication (15 min prior to sacrifice) did not affect NO-Hb levels. These results demonstrate that NAC administration can modulate the massive NO production induced by LPS. This can be attributed mostly to the inhibitory effect of NAC on one of the events leading to iNOS protein expression. This hypothesis is also supported by the lack of effect of late NAC administration.

Oxygen disruption of the 2[4Fe-4S] clusters in Clostridium pasteurianum ferredoxin shown by 1H-NMR.

The ferredoxin from Clostridium pasteurianum, which contains two [4Fe-4S] clusters, was investigated in its oxidized and reduced states by two-dimensional (2D) (1)H-(1)H nuclear Overhauser enhancement spectroscopy (NOESY). Comparison of the data from the oxidized ferredoxin with those published previously revealed the same NOE connectivities. No previous (1)H-(1)H NOESY study of the fully reduced ferredoxin has previously been published. However, it was possible to compare our results with those of a 2D exchange spectroscopy investigation of half-reduced C. pasteurianum ferredoxin. The present results with reduced C. pasteurianum ferredoxin confirm many of the (1)H peaks and NOE interactions reported earlier, revise others, and locate resonances previously undetected. When the ferredoxin was slightly exposed to oxygen, several of the hyperfine shifted resonances were irreversibly influenced. A resonance at 34 ppm in the (1)H NMR spectra of both redox states is indicative of oxygen exposure. These results indicate the importance of keeping the ferredoxin strictly anaerobic during purification and solvent exchange.

Characterization of the intramolecular electron transfer pathway from 2-hydroxyphenazine to the heterodisulfide reductase from Methanosarcina thermophila.

Heterodisulfide reductase (HDR) is a component of the energy-conserving electron transfer system in methanogens. HDR catalyzes the two-electron reduction of coenzyme B-S-S-coenzyme M (CoB-S-S-CoM), the heterodisulfide product of the methyl-CoM reductase reaction, to free thiols, HS-CoB and HS-CoM. HDR from Methanosarcina thermophila contains two b-hemes and two [Fe(4)S(4)] clusters. The physiological electron donor for HDR appears to be methanophenazine (MPhen), a membrane-bound cofactor, which can be replaced by a water-soluble analog, 2-hydroxyphenazine (HPhen). This report describes the electron transfer pathway from reduced HPhen (HPhenH(2)) to CoB-S-S-CoM. Steady-state kinetic studies indicate a ping-pong mechanism for heterodisulfide reduction by HPhenH(2) with the following values: k(cat) = 74 s(-1) at 25 degrees C, K(m) (HPhenH(2)) = 92 microm, K(m) (CoB-S-S-CoM) = 144 microm. Rapid freeze-quench EPR and stopped-flow kinetic studies and inhibition experiments using CO and diphenylene iodonium indicate that only the low spin heme and the high potential FeS cluster are involved in CoB-S-S-CoM reduction by HPhenH(2). Fe-S cluster disruption by mersalyl acid inhibits heme reduction by HPhenH(2), suggesting that a 4Fe cluster is the initial electron acceptor from HPhenH(2). We propose the following electron transfer pathway: HPhenH(2) to the high potential 4Fe cluster, to the low potential heme, and finally, to CoB-S-S-CoM.

Oxalomalate, a competitive inhibitor of aconitase, modulates the RNA-binding activity of iron-regulatory proteins.

We investigated the effect of oxalomalate (OMA, alpha-hydroxy-beta-oxalosuccinic acid), a competitive inhibitor of aconitase, on the RNA-binding activity of the iron-regulatory proteins (IRP1 and IRP2) that control the post-transcriptional expression of various proteins involved in iron metabolism. The RNA-binding activity of IRP was evaluated by electrophoretic mobility-shift assay of cell lysates from 3T3-L1 mouse fibroblasts, SH-SY5Y human cells and mouse livers incubated in vitro with OMA, with and without 2-mercaptoethanol (2-ME). Analogous experiments were performed in vivo by prolonged incubation (72 h) of 3T3-L1 cells with OMA, and by injecting young mice with equimolar concentrations of oxaloacetate and glyoxylate, which are the precursors of OMA synthesis. OMA remarkably decreased the binding activity of IRP1 and, when present, of IRP2, in all samples analysed. In addition, the recovery of IRP1 by 2-ME in the presence of OMA was constantly lower versus control values. These findings suggest that the severe decrease in IRP1 RNA-binding activity depends on: (i) linking of OMA to the active site of aconitase, which prevents the switch to IRP1 and explains resistance to the reducing agents, and (ii) possible interaction of OMA with some functional amino acid residues in IRP that are responsible for binding to the specific mRNA sequences involved in the regulation of iron metabolism.

Stabilization of iron regulatory protein 2, IRP2, by aluminum.

Iron regulatory protein 2 (IRP2) is one of the central regulators of iron homeostasis. IRP2 regulates expression of molecules involved in iron metabolism by binding to iron responsive elements (IREs) in the transcripts of those molecules in iron depletion. IRP2 is regulated by the accelerated degradation initiated by the iron-catalyzed oxidation. Here we report that aluminum antagonizes the iron-induced decrease in IRE binding activity of IRP2. Aluminum also inhibits iron-induced oxidation of IRP2 in vitro. These results suggest that aluminum stabilizes IRP2 by interfering with the iron-catalyzed oxidation, which results in perturbation of iron metabolism.

Iron regulatory element and internal loop/bulge structure for ferritin mRNA studied by cobalt(III) hexammine binding, molecular modeling, and NMR spectroscopy.

The ferritin IRE, a highly conserved (96-99% in vertebrates) mRNA translation regulatory element in animal mRNA, was studied by molecular modeling (using MC-SYM and DOCKING) and by NMR spectroscopy. Cobalt(III) hexammine was used to model hydrated Mg2+. IRE isoforms in other mRNAs regulate mRNA translation or stability; all IREs bind IRPs (iron regulatory proteins). A G.C base pair, conserved in ferritin IREs, spans an internal loop/bulge in the middle of an A-helix and, combined with a dynamic G.U base pair, formed a pocket suitable for Co(III) hexammine binding. On the basis of the effects of Co(III) hexammine on the 1H NMR spectrum and results of automatic docking into the IRE model, the IRE bound Co(III) hexammine at the pocket in the major groove; Mg2+ may bind to the IRE at the same site on the basis of an analogy to Co(III) hexammine and on the Mg2+ inhibition of Cu-(phen)2 cleavage at the site. Distortion of the IRE helix by the internal loop/bulge near a conserved unpaired C required for IRP binding and adjacent to an IRP cross-linking site suggests a role for the pocket in ferritin IRE/IRP interactions.