Cytosolic Fe-S Cluster Protein Maturation and Iron Regulation Are Independent of the Mitochondrial Erv1/Mia40 Import System

The sulfhydryl oxidase Erv1 partners with the oxidoreductase Mia40 to import cysteine-rich proteins in the mitochondrial intermembrane space. In Saccharomyces cerevisiae, Erv1 has also been implicated in cytosolic Fe-S protein maturation and iron regulation. To investigate the connection between Erv1/Mia40-dependent mitochondrial protein import and cytosolic Fe-S cluster assembly, we measured Mia40 oxidation and Fe-S enzyme activities in several erv1 and mia40 mutants. Although all the erv1 and mia40 mutants exhibited defects in Mia40 oxidation, only one erv1 mutant strain (erv1-1) had significantly decreased activities of cytosolic Fe-S enzymes. Further analysis of erv1-1 revealed that it had strongly decreased glutathione (GSH) levels, caused by an additional mutation in the gene encoding the glutathione biosynthesis enzyme glutamate cysteine ligase (GSH1). To address whether Erv1 or Mia40 plays a role in iron regulation, we measured iron-dependent expression of Aft1/2-regulated genes and mitochondrial iron accumulation in erv1 and mia40 strains. The only strain to exhibit iron misregulation is the GSH-deficient erv1-1 strain, which is rescued with addition of GSH. Together, these results confirm that GSH is critical for cytosolic Fe-S protein biogenesis and iron regulation, whereas ruling out significant roles for Erv1 or Mia40 in these pathways.     

Hepatocyte DNA replication in growing liver requires either glutathione or a single allele of txnrd1

Ribonucleotide reductase (RNR) activity requires an electron donor, which in bacteria, yeast, and plants is usually either reduced thioredoxin (Trx) or reduced glutaredoxin. Mice lacking glutathione reductase are viable and, although mice lacking thioredoxin reductase 1 (TrxR1) are embryonic-lethal, several studies have shown that mouse cells lacking the txnrd1 gene, encoding TrxR1, can proliferate normally. To better understand the in vivo electron donor requirements for mammalian RNR, we here investigated whether replication of TrxR1-deficient hepatocytes in mouse livers either employed an alternative source of Trx-reducing activity or, instead, solely relied upon the glutathione (GSH) pathway. Neither normal nor genetically TrxR1-deficient livers expressed substantial levels of mRNA splice forms encoding cytosolic variants of TrxR2, and the TrxR1-deficient livers showed severely diminished total TrxR activity, making it unlikely that any alternative TrxR enzyme activities complemented the genetic TrxR1 deficiency. To test whether the GSH pathway was required for replication, GSH levels were depleted by administration of buthionine sulfoximine (BSO) to juvenile mice. In controls not receiving BSO, replicative indexes were similar in hepatocytes having two, one, or no functional alleles of txnrd1. After BSO treatment, hepatocytes containing either two or one copies of this gene were also normal. However, hepatocytes completely lacking a functional txnrd1 gene exhibited severely reduced replicative indexes after GSH depletion. We conclude that hepatocyte proliferation in vivo requires either GSH or at least one functional allele of txnrd1, demonstrating that either the GSH- or the TrxR1-dependent redox pathway can independently support hepatocyte proliferation during liver growth.     

Cooperative action of antioxidant defense systems in Drosophila

Molecular oxygen is key to aerobic life but is also converted into cytotoxic byproducts referred to as reactive oxygen species (ROS). Intracellular defense systems that protect cells from ROS-induced damage include glutathione reductase (GR), thioredoxin reductase (TrxR), superoxide dismutase (Sod), and catalase (Cat). Sod and Cat constitute an evolutionary conserved ROS defense system against superoxide; Sod converts superoxide anions to H(2)O(2), and Cat prevents free hydroxyl radical formation by breaking down H(2)O(2) into oxygen and water. As a consequence, they are important effectors in the life span determination of the fly Drosophila. ROS defense by TrxR and GR is more indirect. They transfer reducing equivalents from NADPH to thioredoxin (Trx) and glutathione disulfide (GSSG), respectively, resulting in Trx(SH)(2) and glutathione (GSH), which act as effective intracellular antioxidants. TrxR and GR were found to be molecularly conserved. However, the single GR homolog of Drosophila specifies TrxR activity, which compensates for the absence of a true GR system for recycling GSH. We show that TrxR null mutations reduce the capacity to adequately protect cells from cytotoxic damage, resulting in larval death, whereas mutations causing reduced TrxR activity affect pupal eclosion and cause a severe reduction of the adult life span. We also provide genetic evidence for a functional interaction between TrxR, Sod1, and Cat, indicating that the burden of ROS metabolism in Drosophila is shared by the two defense systems.     

Exacerbation of copper toxicity in primary neuronal cultures depleted of cellular glutathione

Perturbations to glutathione (GSH) metabolism may play an important role in neurodegenerative disorders such as Alzheimer's, Parkinson's, and prion diseases. A primary function of GSH is to prevent the toxic interaction between free radicals and reactive transition metals such as copper (Cu). Due to the potential role of Cu in neurodegeneration, we examined the effect of GSH depletion on Cu toxicity in murine primary neuronal cultures. Depletion of cellular GSH with L-buthionine-[S,R]-sulfoximine resulted in a dramatic potentiation of Cu toxicity in neurons without effect on iron (Fe) toxicity. Similarly, inhibition of glutathione reductase (GR) activity with 1,3-bis(2-chloroethyl)-1-nitrosurea also increased Cu toxicity in neurons. To determine if the Alzheimer's amyloid-beta (Abeta) peptide can affect neuronal resistance to transition metal toxicity, we exposed cultures to nontoxic concentrations of Abeta25-35 in the presence or absence of Cu or Fe. Abeta25-35 pretreatment was found to deplete neuronal GSH and increase GR activity, confirming the ability of Abeta to perturb neuronal GSH homeostasis. Abeta25-35 pretreatment potently increased Cu toxicity but had no effect on Fe toxicity. These studies demonstrate an important role for neuronal GSH homeostasis in selective protection against Cu toxicity, a finding with widespread implications for neurodegenerative disorders.     

Cadmium induced mitochondrial redox changes in germinating pea seed

Mitochondria play an essential role in producing the energy required for seedling growth following imbibition. Heavy metals, such as cadmium impair mitochondrial functioning in part by altering redox regulation. The activities of two protein redox systems present in mitochondria, thioredoxin (Trx) and glutaredoxin (Grx), were analysed in the cotyledons and embryo of pea (Pisum sativum L.) germinating seeds exposed to toxic Cd concentration. Compared to controls, Cd-treated germinating seeds showed a decrease in total soluble protein content, but an increase in –SH content. Under Cd stress conditions, Grx and glutathione reductase (GR) activities as well as glutathione (GSH) concentrations decreased both in cotyledons and the embryo. Similar results were obtained with the Trx system: Trx and NADPH-dependent thioredoxin reductase (NTR) activities were not stimulated, whereas total NAD(P) contents diminished in the embryo. However, Cd enhanced the levels of all components of the Trx system in the cotyledons. On the other hand, Cd caused a significant increase in oxidative stress parameters such as the redox ratio of coenzymes (oxidized to reduced forms) and NAD(P)H oxidase activities. These results indicate that Cd induces differential redox responses on different seed tissues. We suggest that neither Grx system nor Trx one may improve the redox status of mitochondrial thiols in the embryo of germinating pea seeds exposed to Cd toxicity, but in the cotyledons the contribution of Trx/NTR/NADPH can be established in despite the vulnerability of the coenzyme pools due to enzymatic oxidation.     

Gpx1 is a stationary phase-specific thioredoxin peroxidase in fission yeast

The genome sequence of Schizosaccharomyces pombe reveals only one gene for a putative glutathione peroxidase (gpx1⁺). The Gpx1 protein has a peroxidase activity but preferred thioredoxin to glutathione as an electron donor when examined in vitro and in vivo, and therefore is a thioredoxin peroxidase. Besides H₂O₂, it can reduce alkyl and phospholipid hydroperoxides. Expression of the gpx1 gene was elevated at the stationary phase, and we found that it supported long-term survival of S. pombe. The mutant also exhibited some defect in the activity of aconitase, an oxidation-labile Fe-S enzyme in mitochondria. Activity of sulfite reductase, a labile Fe-S enzyme in the cytosol, was also dramatically lowered in the mutant in the stationary phase. The Gpx1 protein, without any obvious targeting sequence, was localized in mitochondria as well as in the cytosol. Therefore, Gpx1 must serve to ensure optimal mitochondrial function and cytosolic environment, especially in the stationary phase.     

Platyhelminth mitochondrial and cytosolic redox homeostasis is controlled by a single thioredoxin glutathione reductase and dependent on selenium and glutathione

Platyhelminth parasites are a major health problem in developing countries. In contrast to their mammalian hosts, platyhelminth thiol-disulfide redox homeostasis relies on linked thioredoxin-glutathione systems, which are fully dependent on thioredoxin-glutathione reductase (TGR), a promising drug target. TGR is a homodimeric enzyme comprising a glutaredoxin domain and thioredoxin reductase (TR) domains with a C-terminal redox center containing selenocysteine (Sec). In this study, we demonstrate the existence of functional linked thioredoxin-glutathione systems in the cytosolic and mitochondrial compartments of Echinococcus granulosus, the platyhelminth responsible for hydatid disease. The glutathione reductase (GR) activity of TGR exhibited hysteretic behavior regulated by the [GSSG]/[GSH] ratio. This behavior was associated with glutathionylation by GSSG and abolished by deglutathionylation. The K(m) and k(cat) values for mitochondrial and cytosolic thioredoxins (9.5 microm and 131 s(-1), 34 microm and 197 s(-1), respectively) were higher than those reported for mammalian TRs. Analysis of TGR mutants revealed that the glutaredoxin domain is required for the GR activity but did not affect the TR activity. In contrast, both GR and TR activities were dependent on the Sec-containing redox center. The activity loss caused by the Sec-to-Cys mutation could be partially compensated by a Cys-to-Sec mutation of the neighboring residue, indicating that Sec can support catalysis at this alternative position. Consistent with the essential role of TGR in redox control, 2.5 microm auranofin, a known TGR inhibitor, killed larval worms in vitro. These studies establish the selenium- and glutathione-dependent regulation of cytosolic and mitochondrial redox homeostasis through a single TGR enzyme in platyhelminths.     

Antioxidant defences and homeostasis of reactive oxygen species in different human mitochondrial DNA-depleted cell lines.

Three pairs of parental (rho+) and established mitochondrial DNA depleted (rho0) cells, derived from bone, lung and muscle were used to verify the influence of the nuclear background and the lack of efficient mitochondrial respiratory chain on antioxidant defences and homeostasis of intracellular reactive oxygen species (ROS). Mitochondrial DNA depletion significantly lowered glutathione reductase activity, glutathione (GSH) content, and consistently altered the GSH2 : oxidized glutathione ratio in all of the rho0 cell lines, albeit to differing extents, indicating the most oxidized redox state in bone rho0 cells. Activity, as well as gene expression and protein content, of superoxide dismutase showed a decrease in bone and muscle rho0 cell lines but not in lung rho0 cells. GSH peroxidase activity was four times higher in all three rho0 cell lines in comparison to the parental rho+, suggesting that this may be a necessary adaptation for survival without a functional respiratory chain. Taken together, these data suggest that the lack of respiratory chain prompts the cells to reduce their need for antioxidant defences in a tissue-specific manner, exposing them to a major risk of oxidative injury. In fact bone-derived rho0 cells displayed the highest steady-state level of intracellular ROS (measured directly by 2',7'-dichlorofluorescin, or indirectly by aconitase activity) compared to all the other rho+ and rho0 cells, both in the presence or absence of glucose. Analysis of mitochondrial and cytosolic/iron regulatory protein-1 aconitase indicated that most ROS of bone rho0 cells originate from sources other than mitochondria.     

Loss of Cardiolipin Leads to Perturbation of Mitochondrial and Cellular Iron Homeostasis

Cardiolipin (CL) is the signature phospholipid of mitochondrial membranes, where it is synthesized locally and plays a critical role in mitochondrial bioenergetic functions. The importance of CL in human health is underscored by the observation that perturbation of CL biosynthesis causes the severe genetic disorder Barth syndrome. To fully understand the cellular response to the loss of CL, we carried out genome-wide expression profiling of the yeast CL mutant crd1Δ. Our results show that the loss of CL in this mutant leads to increased expression of iron uptake genes accompanied by elevated levels of mitochondrial iron and increased sensitivity to iron and hydrogen peroxide. Previous studies have shown that increased mitochondrial iron levels result from perturbations in iron-sulfur (Fe-S) cluster biogenesis. Consistent with an Fe-S defect, deletion of ISU1, one of two ISU genes that encode the mitochondrial Fe-S scaffolding protein essential for the synthesis of Fe-S clusters, led to synthetic growth defects with the crd1Δ mutant. We further show that crd1Δ cells have reduced activities of mitochondrial Fe-S enzymes (aconitase, succinate dehydrogenase, and ubiquinol-cytochrome c oxidoreductase), as well as cytosolic Fe-S enzymes (sulfite reductase and isopropylmalate isomerase). Increased expression of ATM1 or YAP1 did not rescue the Fe-S defects in crd1Δ. These findings show for the first time that CL is required for Fe-S biogenesis to maintain mitochondrial and cellular iron homeostasis.     

The role of mitochondrial glutathione and cellular protein sulfhydryls in formaldehyde toxicity in glutathione-depleted rat hepatocytes

Depletion of cellular GSH by diethyl maleate (DEM) potentiates CH2O toxicity in isolated rat hepatocytes and it was postulated that this increase in toxicity is due to the further decrease in GSH caused by CH2O in DEM-pretreated hepatocytes (1). The present investigation was conducted to investigate further the effects of CH2O, DEM, and acrolein (a compound which is structurally related to CH2O and DEM) on subcellular GSH pools and on protein sulfhydryl groups (PSH). CH2O caused a decrease in cytosolic GSH but had no effect on mitochondrial GSH either in previously untreated hepatocytes or in DEM-pretreated hepatocytes in which GSH was approximately 25% of control. DEM decreased both cytosolic and mitochondrial GSH but it did not produce toxicity. Neither CH2O (up to 7.5 mM) nor DEM (20 mM) decreased PSH. However, in cells pretreated with 1 mM DEM, CH2O (7.5 mM) decreased PSH and this effect preceded cell death. Acrolein decreased both cytosolic and mitochondrial GSH and it also decreased PSH significantly prior to causing cell death. CH2O and acrolein stimulated phosphorylase alpha activity, indicative of an increase in cytosolic free Ca2+, by a PSH-independent and PSH-dependent mechanism, respectively. These results suggest that the further depletion of cellular GSH by CH2O in DEM-pretreated cells is not due to the depletion of mitochondrial GSH. CH2O toxicity in DEM-pretreated cells is, however, correlated with depletion of PSH. The critical sulfhydryl protein(s) responsible for cell death remain to be more clearly defined.     

Glutathione and Glutaredoxin Act as a Backup of Human Thioredoxin Reductase 1 to Reduce Thioredoxin 1 Preventing Cell Death by Aurothioglucose

Thioredoxin reductase 1 (TrxR1) in cytosol is the only known reductant of oxidized thioredoxin 1 (Trx1) in vivo so far. We and others found that aurothioglucose (ATG), a well known active-site inhibitor of TrxR1, inhibited TrxR1 activity in HeLa cell cytosol but had no effect on the viability of the cells. Using a redox Western blot analysis, no change was observed in redox state of Trx1, which was mainly fully reduced with five sulfhydryl groups. In contrast, auranofin killed cells and oxidized Trx1, also targeting mitochondrial TrxR2 and Trx2. Combining ATG with ebselen gave a strong synergistic effect, leading to Trx1 oxidation, reactive oxygen species accumulation, and cell death. We hypothesized that there should exist a backup system to reduce Trx1 when only TrxR1 activity was lost. Our results showed that physiological concentrations of glutathione, NADPH, and glutathione reductase reduced Trx1 in vitro and that the reaction was strongly stimulated by glutaredoxin1. Simultaneous depletion of TrxR activity by ATG and glutathione by buthionine sulfoximine led to overoxidation of Trx1 and loss of HeLa cell viability. In conclusion, the glutaredoxin system and glutathione have a backup role to keep Trx1 reduced in cells with loss of TrxR1 activity. Monitoring the redox state of Trx1 shows that cell death occurs when Trx1 is oxidized, followed by general protein oxidation catalyzed by the disulfide form of thioredoxin.     

Adaptation of subcellular glutathione detoxification system to stress conditions in choline-deficient diet induced rat fatty liver

The response of fatty liver to stress conditions (t-butyl hydroperoxide [t-BH] or 36 h of fasting) was investigated by assessing intracellular glutathione (GSH) compartmentation and redox status, GSH peroxidase (GSH-Px) and reductase (GSSG-Rx) activities, lipid peroxidation (TBARs) and serum ALT levels in rats on a choline-deficient diet. Baseline cytosolic GSH was similar between fatty and normal livers, while the mitochondrial GSH content was significantly lower in fatty livers. With the except of cytosolic GSH-Px activity, steatosis was associated with significantly higher GSH-related enzymes activities. Liver TBARs and serum ALT levels were also higher. Administration of t-BH significantly decreased the concentration of cytosolic GSH, increased GSSG levels in all the compartments, and increased TBARs levels in cytosol and mitochondria and serum ALT; all these alterations were more marked in rats with fatty liver. Fasting decreased the concentration of GSH in all the compartments both in normal and fatty livers, increased GSSG, TBARs and ALT levels, and decreased by 50% the activities of GSH-related enzymes. Administration of diethylmaleimide (DEM) resulted in cytosolic and microsomal GSH pool depletion. Administration of t-BH to DEM-treated rats further affected cytosolic GSH and enhanced ALT levels, whereas the application of fasting to GSH depleted rats mainly altered the mitochondrial GSH system, especially in fatty livers. This study shows that fatty livers have a weak compensation of hepatic GSH regulation, which fails under stress conditions, thus increasing the fatty liver's susceptibility to oxidative damage. Differences emerge among subcellular compartments which point to differential adaptation of these organelles to fatty degeneration.     

Both human ferredoxins 1 and 2 and ferredoxin reductase are important for iron-sulfur cluster biogenesis

Ferredoxins are iron-sulfur proteins that have been studied for decades because of their role in facilitating the monooxygenase reactions catalyzed by p450 enzymes. More recently, studies in bacteria and yeast have demonstrated important roles for ferredoxin and ferredoxin reductase in iron-sulfur cluster assembly. The human genome contains two homologous ferredoxins, ferredoxin 1 (FDX1) and ferredoxin 2 (FDX2--formerly known as ferredoxin 1L). More recently, the roles of these two human ferredoxins in iron-sulfur cluster assembly were assessed, and it was concluded that FDX1 was important solely for its interaction with p450 enzymes to synthesize mitochondrial steroid precursors, whereas FDX2 was used for synthesis of iron-sulfur clusters, but not steroidogenesis. To further assess the role of the FDX-FDXR system in mammalian iron-sulfur cluster biogenesis, we performed siRNA studies on FDX1 and FDX2, on several human cell lines, using oligonucleotides identical to those previously used, along with new oligonucleotides that specifically targeted each gene. We concluded that both FDX1 and FDX2 were important in iron-sulfur cluster biogenesis. Loss of FDX1 activity disrupted activity of iron-sulfur cluster enzymes and cellular iron homeostasis, causing mitochondrial iron overload and cytosolic iron depletion. Moreover, knockdown of the sole human ferredoxin reductase, FDXR, diminished iron-sulfur cluster assembly and caused mitochondrial iron overload in conjunction with cytosolic depletion. Our studies suggest that interference with any of the three related genes, FDX1, FDX2 or FDXR, disrupts iron-sulfur cluster assembly and maintenance of normal cytosolic and mitochondrial iron homeostasis.     

Na-K-ATPase in rat cerebellar granule cells is redox sensitive

Redox-induced regulation of the Na-K-ATPase was studied in dispersed rat cerebellar granule cells. Intracellular thiol redox state was modulated using glutathione (GSH)-conjugating agents and membrane-permeable ethyl ester of GSH (et-GSH) and Na-K-ATPase transport and hydrolytic activity monitored as a function of intracellular reduced thiol concentration. Depletion of cytosolic and mitochondrial GSH pools caused an increase in free radical production in mitochondria and rapid ATP deprivation with a subsequent decrease in transport but not hydrolytic activity of the Na-K-ATPase. Selective conjugation of cytosolic GSH did not affect free radical production and Na-K-ATPase function. Unexpectedly, overloading of cerebellar granule cells with GSH triggered global free radical burst originating most probably from GSH autooxidation. The latter was not followed by ATP depletion but resulted in suppression of active K(+) influx and a modest increase in mortality. Suppression of transport activity of the Na-K-ATPase was observed in granule cells exposed to both permeable et-GSH and impermeable GSH, with inhibitory effects of external and cytosolic GSH being additive. The obtained data indicate that redox state is a potent regulator of the Na-K-ATPase function. Shifts from an "optimal redox potential range" to higher or lower levels cause suppression of the Na-K pump activity.     

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.     

Redox-sensitive YFP sensors monitor dynamic nuclear and cytosolic glutathione redox changes

Intracellular redox homeostasis is crucial for many cellular functions but accurate measurements of cellular compartment-specific redox states remain technically challenging. To better characterize redox control in the nucleus, we targeted a yellow fluorescent protein-based redox sensor (rxYFP) to the nucleus of the yeast Saccharomyces cerevisiae. Parallel analyses of the redox state of nucleus-rxYFP and cytosol-rxYFP allowed us to monitor distinctively dynamic glutathione (GSH) redox changes within these two compartments under a given condition. We observed that the nuclear GSH redox environment is highly reducing and similar to the cytosol under steady-state conditions. Furthermore, these sensors are able to detect redox variations specific for their respective compartments in glutathione reductase (Glr1) and thioredoxin pathway (Trr1, Trx1, Trx2) mutants that have altered subcellular redox environments. Our mutant redox data provide in vivo evidence that glutathione and the thioredoxin redox systems have distinct but overlapping functions in controlling subcellular redox environments. We also monitored the dynamic response of nucleus-rxYFP and cytosol-rxYFP to GSH depletion and to exogenous low and high doses of H₂O₂ bursts. These observations indicate a rapid and almost simultaneous oxidation of both nucleus-rxYFP and cytosol-rxYFP, highlighting the robustness of the rxYFP sensors in measuring real-time compartmental redox changes. Taken together, our data suggest that the highly reduced yeast nuclear and cytosolic redox states are maintained independently to some extent and under distinct but subtle redox regulation. Nucleus- and cytosol-rxYFP register compartment-specific localized redox fluctuations that may involve exchange of reduced and/or oxidized glutathione between these two compartments. Finally, we confirmed that GSH depletion has profound effects on mitochondrial genome stability but little effect on nuclear genome stability, thereby emphasizing that the critical requirement for GSH during growth is linked to a mitochondria-dependent process.