Methylglyoxal-Scavenging Enzyme Activities Trigger Erythroascorbate Peroxidase and Cytochrome c Peroxidase in Glutathione-Depleted Candida albicans.

γ-Glutamylcysteine synthetase (Gcs1) and glutathione reductase (Glr1) activity maintains minimal levels of cellular methylglyoxal in Candida albicans. In glutathione-depleted Δgcs1, we previously saw that NAD(H)-linked methylglyoxal oxidoreductase (Mgd1) and alcohol dehydrogenase (Adh1) are the most active methylglyoxal scavengers. With methylglyoxal accumulation, disruptants lacking MGD1 or ADH1 exhibit a poor redox state. However, there is little convincing evidence for a reciprocal relationship between methylglyoxal scavenger genes-disrupted mutants and changes in glutathione-(in)dependent redox regulation. Herein, we attempt to demonstrate a functional role for methylglyoxal scavengers, modeled on a triple disruptant (Δmgd1/Δadh1/Δgcs1), to link between antioxidative enzyme activities and their metabolites in glutathione-depleted conditions. Despite seeing elevated methylglyoxal in all of the disruptants, the result saw a decrease in pyruvate content in Δmgd1/Δadh1/Δgcs1 which was not observed in double gene-disrupted strains such as Δmgd1/Δgcs1 and Δadh1/Δgcs1. Interestingly, Δmgd1/Δadh1/Δgcs1 exhibited a significantly decrease in H2O2 and superoxide which was also unobserved in Δmgd1/Δgcs1 and Δadh1/Δgcs1. The activities of the antioxidative enzymes erythroascorbate peroxidase and cytochrome c peroxidase were noticeably higher in Δmgd1/Δadh1/Δgcs1 than in the other disruptants. Meanwhile, Glr1 activity severely diminished in Δmgd1/Δadh1/Δgcs1. Monitoring complementary gene transcripts between double gene-disrupted Δmgd1/Δgcs1 and Δadh1/Δgcs1 supported the concept of an unbalanced redox state independent of the Glr1 activity for Δmgd1/Δadh1/Δgcs1. Our data demonstrate the reciprocal use of Eapx1 and Ccp1 in the absence of both methylglyoxal scavengers; that being pivotal for viability in non-filamentous budding yeast.

Alcohol dehydrogenase 1 and NAD(H)-linked methylglyoxal oxidoreductase reciprocally regulate glutathione-dependent enzyme activities in Candida albicans.

Glutathione reductase (Glr1) activity controls cellular glutathione and reactive oxygen species (ROS). We previously demonstrated two predominant methylglyoxal scavengers-NAD(H)-linked methylglyoxal oxidoreductase (Mgd1) and alcohol dehydrogenase 1 (Adh1)-in glutathione-depleted γ-glutamyl cysteinyl synthetase-disrupted Candida albicans. However, experimental evidence for Candida pathophysiology lacking the enzyme activities of Mgd1 and Adh1 on glutathione-dependent redox regulation remains unclear. Herein, we have aimed to demonstrate that glutathione-dependent enzyme activities coupled with cellular ROS changes is regulated by methylglyoxal accumulation in Δmgd1/Δadh1 double disruptants. Δmgd1/Δadh1 showed severe growth defects and G1-phase cell cycle arrest. The observed complementary and reciprocal methylglyoxal-oxidizing and methylglyoxalreducing activities between Δmgd1 and Δadh1 were not always exhibited in Δmgd1/Δadh1. Although intracellular accumulation of methylglyoxal and pyruvate was shown in all disruptants, to a greater or lesser degree, methylglyoxal was particularly accumulated in the Δmgd1/Δadh1 double disruptant. While cellular ROS significantly increased in Δmgd1 and Δadh1 as compared to the wild-type, Δmgd1/Δadh1 underwent a decrease in ROS in contrast to Δadh1. Despite the experimental findings underlining the importance of the undergoing unbalanced redox state of Δmgd1/Δadh1, glutathione-independent antioxidative enzyme activities did not change during proliferation and filamentation. Contrary to the significantly lowered glutathione content and Glr1 enzyme activity, the activity staining-based glutathione peroxidase activities concomitantly increased in this mutant. Additionally, the enhanced GLR1 transcript supported our results in Δmgd1/Δadh1, indicating that deficiencies of both Adh1 and Mgd1 activities stimulate specific glutathione-dependent enzyme activities. This suggests that glutathione-dependent redox regulation is evidently linked to C. albicans pathogenicity under the control of methylglyoxal-scavenging activities.

Anti-σ factor YlaD regulates transcriptional activity of σ factor YlaC and sporulation via manganese-dependent redox-sensing molecular switch in Bacillus subtilis.

YlaD, a membrane-anchored anti-sigma (σ) factor of Bacillus subtilis, contains a HX3CXXC motif that functions as a redox-sensing domain and belongs to one of the zinc (Zn)-co-ordinated anti-σ factor families. Despite previously showing that the YlaC transcription is controlled by YlaD, experimental evidence of how the YlaC-YlaD interaction is affected by active cysteines and/or metal ions is lacking. Here, we showed that the P yla promoter is autoregulated solely by YlaC. Moreover, reduced YlaD contained Zn and iron, while oxidized YlaD did not. Cysteine substitution in YlaD led to changes in its secondary structure; Cys3 had important structural functions in YlaD, and its mutation caused dissociation from YlaC, indicating the essential requirement of a HX3CXXC motif for regulating interactions of YlaC with YlaD. Analyses of the far-UV CD spectrum and metal content revealed that the addition of Mn ions to Zn-YlaD changed its secondary structure and that iron was substituted for manganese (Mn). The ylaC gene expression using ßGlu activity from P yla :gusA was observed at the late-exponential and early-stationary phase, and the ylaC-overexpressing mutant constitutively expressed gene transcripts of clpP and sigH, an important alternative σ factor regulated by ClpXP. Collectively, our data demonstrated that YlaD senses redox changes and elicits increase in Mn ion concentrations and that, in turn, YlaD-mediated transcriptional activity of YlaC regulates sporulation initiation under oxidative stress and Mn-substituted conditions by regulating clpP gene transcripts. This is the first report of the involvement of oxidative stress-responsive B. subtilis extracytoplasmic function σ factors during sporulation via a Mn-dependent redox-sensing molecular switch.

Inducible NAD(H)-linked methylglyoxal oxidoreductase regulates cellular methylglyoxal and pyruvate through enhanced activities of alcohol dehydrogenase and methylglyoxal-oxidizing enzymes in glutathione-depleted Candida albicans.

BACKGROUND: High methylglyoxal content disrupts cell physiology, but mammals have scavengers to prevent glycolytic and mitochondrial dysfunctions. In yeast, methylglyoxal accumulation triggers methylglyoxal-oxidizing alcohol dehydrogenase (Adh1) activity. While methylglyoxal reductases and glyoxalases have been well studied in prokaryotes and eukaryotes, experimental evidence for methylglyoxal dehydrogenase (Mgd) and other catalytic activities of this enzyme affecting glycolysis and the tricarboxylic acid cycle is lacking. METHODS: A glycine-rich cytoplasmic Mgd protein, designated as Mgd1/Grp2, was isolated from glutathione-depleted Candida albicans. The effects of Mgd1/Grp2 activities on metabolic pathophysiology were investigated using knockout and overexpression mutants. We measured glutathione-(in)dependent metabolite contents and metabolic effects, including viability, oxygen consumption, ADH1 transcripts, and glutathione reductase and α-ketoglutarate dehydrogenase activities in the mutants. Based on the findings, methylglyoxal-oxidizing proteins were monitored to determine effects of MGD1/GRP2 disruption on methylglyoxal-scavenging traits during glutathione deprivation. RESULTS: Methylglyoxal-oxidizing NAD(H)-linked Mgd1/Grp2 was found solely in glutathione auxotrophs, and it catalyzed the reduction of both methylglyoxal and pyruvate. MGD1/GRP2 disruptants showed growth defects, cell-cycle arrest, and methylglyoxal and pyruvate accumulation with mitochondrial impairment, regardless of ADH1 compensation. Other methylglyoxal-oxidizing enzymes were identified as key glycolytic enzymes with enhanced activity and transcription in MGD1/GRP2 disruptants, irrespective of glutathione content. CONCLUSIONS: Failure of methylglyoxal and pyruvate dissimilation by Mgd1/Grp2 deficiency leads to poor glutathione-dependent redox regulation despite compensation by Adh1. GENERAL SIGNIFICANCE: This is the first report that multifunctional Mgd activities contribute to scavenging methylglyoxal and pyruvate to maintain metabolic homeostasis and the redox pool via glycolytic enzymes and Adh1 expression.

Cytochrome c peroxidase regulates intracellular reactive oxygen species and methylglyoxal via enzyme activities of erythroascorbate peroxidase and glutathione-related enzymes in Candida albicans.

D-erythroascorbate peroxidase (EAPX1) deficiency causes glutathione deprivation, leading to the accumulation of methylglyoxal and reactive oxygen species (ROS), and especially, induction of cytochrome c peroxidase (Ccp1) in Candida albicans. Nevertheless, reciprocal effects between changes in Ccp1 activity and the antioxidative D-erythroascorbic acid- and glutathione-dependent redox status, which reflects methylglyoxal biosynthesis altering pathophysiology are unclear in eukaryotes. To elucidate the effect of CCP1 expression on EAPX1 and glutathione reductase (Glr1) activity-mediated D-erythroascorbic acid biosynthesis and redox homeostasis, the CCP1 gene was disrupted and overexpressed. First, we demonstrated both glutathione-independent and-dependent metabolite contents and their corresponding gene transcripts and enzyme activities (i.e., Ccp1, catalase-peroxidase [KatG], superoxide dismutase [Sod], Eapx1, and Glr1) in CCP1 mutants. Second, methylglyoxal-oxidizing alcohol dehydrogenase (Adh1) and methylglyoxal-reducing oxidoreductase activity on glycolytic methylglyoxal and pyruvate production and NAD(P)H content were determined in these mutants. Contrary to our expectation, CCP1 disruption (42.19±3.22nmolO2h-1mgwetcell-1) failed to affect cell respiration compared to the wild-type strain (41.62±7.11nmolO2h-1mgwetcell-1) under cyanide treatment, and in contrast to hydrogen peroxide (H2O2) treatment (21.74±1.03nmol O2h-1mgwetcell-1). Additionally, Ccp1 predominantly detoxified H2O2 rather than negligible scavenging activities towards methylglyoxal and other oxidants. CCP1 deficiency stimulated Sod and Adh1 activity but downregulated Glr1, Eapx1, catalase, and peroxidase activity while enhancing KatG, EAPX1, and GLR1 transcription by decreasing glutathione and D-erythroascorbic acid and increasing pyruvate. Noticeably, the ROS-accumulating CCP1-deficient mutant maintained steady-state levels of methylglyoxal, which was revealed to be regulated by methylglyoxal-oxidizing and -reducing activity with drastic changes in NAD(P)H. We confirmed and clarified our results by showing that CCP1/EAPX1 double disruptants underwent severe growth defects due to the D-erythroascorbic acid and glutathione depletion because of pyruvate overaccumulation. These observations were made in both budding and hyphal-growing CCP1 mutants. The revealed metabolic network involving Ccp1 and other redox regulators affected ROS and methylglyoxal through D-erythroascorbic acid and glutathione-dependent metabolites, thereby influencing dimorphism. This is the first report of the Ccp1-mediated D-erythroascorbic acid and glutathione biosynthesis accompanying methylglyoxal scavengers for full fungal virulence.

Methylglyoxal upregulates Dictyostelium discoideum slug migration by triggering glutathione reductase and methylglyoxal reductase activity.

Glutathione (GSH)-deprived Dictyostelium discoideum accumulates methylglyoxal (MG) and reactive oxygen species (ROS) during vegetative growth. However, the reciprocal effects of the production and regulation of these metabolites on differentiation and cell motility are unclear. Based on the inhibitory effects of γ-glutamylcysteine synthetase (gcsA) disruption and GSH reductase (gsr) overexpression on aggregation and culmination, respectively, we overexpressed GSH-related genes encoding superoxide dismutase (Sod2), catalase (CatA), and Gcs, in D. discoideum. Wild-type KAx3 and gcsA-overexpressing (gcsAOE) slugs maintained GSH levels at levels of approximately 2.1-fold less than the reference GSH synthetase-overexpressing mutant; their GSH levels did not correlate with slug migration ability. Through prolonged KAx3 migration by treatment with MG and H2O2, we found that MG increased after the mound stage in this strain, with a 2.6-fold increase compared to early developmental stages; in contrast, ROS were maintained at high levels throughout development. While the migration-defective sod2- and catA-overexpressing mutant slugs (sod2OE and catAOE) decreased ROS levels by 50% and 53%, respectively, these slugs showed moderately decreased MG levels (36.2±5.8 and 40.7±1.6nmolg-1 cells wet weight, P<0.05) compared to the parental strain (54.2±3.5nmolg-1). Importantly, defects in the migration of gcsAOE slugs decreased MG considerably (13.8±4.2nmolg-1, P<0.01) along with a slight decrease in ROS. In contrast to the increase observed in migrating sod2OE and catAOE slugs by treatment with MG and H2O2, the migration of gcsAOE slugs appeared unaffected. This behavior was caused by MG-triggered Gsr and NADPH-linked aldolase reductase activity, suggesting that GSH biosynthesis in gcsAOE slugs is specifically used for MG-scavenging activity. This is the first report showing that MG upregulates slug migration via MG-scavenging-mediated differentiation.

Candida albicans glutathione reductase downregulates Efg1-mediated cyclic AMP/protein kinase A pathway and leads to defective hyphal growth and virulence upon decreased cellular methylglyoxal content accompanied by activating alcohol dehydrogenase and glycolytic enzymes.

BACKGROUND: Glutathione reductase maintains the glutathione level in a reduced state. As previously demonstrated, glutathione is required for cell growth/division and its biosynthesizing-enzyme deficiency causes methylglyoxal accumulation. However, experimental evidences for reciprocal relationships between Cph1-/Efg1-mediated signaling pathway regulation and methylglyoxal production exerted by glutathione reductase on yeast morphology remain unclear. METHODS: Glutathione reductase (GLR1) disruption/overexpression were performed to investigate aspects of pathological/morphological alterations in Candida albicans. These assumptions were proved by observations of cellular susceptibility to oxidants and thiols, and measurements of methylglyoxal and glutathione content in hyphal-inducing conditions mainly through the activity of GLR1-overexpressing cells. Additionally, the transcriptional/translational levels of bioenergetic enzymes and dimorphism-regulating protein kinases were examined in the strain. RESULTS: The GLR1-deficient strain was non-viable when GLR1 expression under the control of a CaMAL2 promoter was conditionally repressed, despite partial rescue of growth by exogenous thiols. During filamentation, non-growing hyphal GLR1-overexpressing cells exhibited resistance against oxidants and cellular methylglyoxal was significantly decreased, which concomitantly increased expressions of genes encoding energy-generating enzymes, including fructose-1,6-bisphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase, and alcohol dehydrogenase (ADH1), with remarkable repression of Efg1-signaling cascades. CONCLUSIONS: This is the first report that GLR1-triggered Efg1-mediated signal transduction repression strictly reduces dimorphic switching and virulence by maintaining the basal level of methylglyoxal following the enhanced gene expressions of glycolytic enzymes and ADH1. GENERAL SIGNIFICANCE: The Efg1 downregulatory mechanism by GLR1 expression has possibilities to involve in other complex network of signal pathways. Understanding how GLR1 overexpression affects multiple signaling pathways can help identify attractive targets for antifungal drugs.

Glutathione upregulates cAMP signalling via G protein alpha 2 during the development of Dictyostelium discoideum.

Despite the importance of glutathione in Dictyostelium, the role of glutathione synthetase (gshB/GSS) has not been clearly investigated. In this study, we observed that increasing glutathione content by constitutive expression of gshB leads to mound-arrest and defects in 3',5'-cyclic adenosine monophosphate (cAMP)-mediated aggregation and developmental gene expression. The overexpression of gpaB encoding G protein alpha 2 (Gα2), an essential component of the cAMP signalling pathway, results in a phenotype similar to that caused by gshB overexpression, whereas gpaB knockdown in gshB-overexpressing cells partially rescues the above-mentioned phenotypic defects. Furthermore, Gα2 is highly enriched at the plasma membrane of gshB-overexpressing cells compared to wild-type cells. Therefore, our findings suggest that glutathione upregulates cAMP signalling via Gα2 modulation during Dictyostelium development.

Activin pathway enhances colorectal cancer stem cell self-renew and tumor progression.

Activin belongs to transforming growth factor (TGF)-ß super family of growth and differentiation factors and activin pathway participated in broad range of cell process. Studies elaborated activin pathway maintain pluripotency in human stem cells and suggest that the function of activin/nodal signaling in self-renew would be conserved across embryonic and adult stem cells. In this study, we tried to determine the effect of activin signaling pathway in regulation of cancer stem cells as a potential target for cancer therapy in clinical trials. A population of colorectal cancer cells was selected by the treatment of activin A. This population of cell possessed stem cell character with sphere formation ability. We demonstrated activin pathway enhanced the colorectal cancer stem cells self-renew and contribute to colorectal cancer progression in vivo. Targeting activin pathway potentially provide effective strategy for colorectal cancer therapy.

Two distinct mechanisms of transcriptional regulation by the redox sensor YodB.

For bacteria, cysteine thiol groups in proteins are commonly used as thiol-based switches for redox sensing to activate specific detoxification pathways and restore the redox balance. Among the known thiol-based regulatory systems, the MarR/DUF24 family regulators have been reported to sense and respond to reactive electrophilic species, including diamide, quinones, and aldehydes, with high specificity. Here, we report that the prototypical regulator YodB of the MarR/DUF24 family from Bacillus subtilis uses two distinct pathways to regulate transcription in response to two reactive electrophilic species (diamide or methyl-p-benzoquinone), as revealed by X-ray crystallography, NMR spectroscopy, and biochemical experiments. Diamide induces structural changes in the YodB dimer by promoting the formation of disulfide bonds, whereas methyl-p-benzoquinone allows the YodB dimer to be dissociated from DNA, with little effect on the YodB dimer. The results indicate that B. subtilis may discriminate toxic quinones, such as methyl-p-benzoquinone, from diamide to efficiently manage multiple oxidative signals. These results also provide evidence that different thiol-reactive compounds induce dissimilar conformational changes in the regulator to trigger the separate regulation of target DNA. This specific control of YodB is dependent upon the type of thiol-reactive compound present, is linked to its direct transcriptional activity, and is important for the survival of B. subtilis This study of B. subtilis YodB also provides a structural basis for the relationship that exists between the ligand-induced conformational changes adopted by the protein and its functional switch.

Polyamines regulate cell growth and cellular methylglyoxal in high-glucose medium independently of intracellular glutathione.

Polyamines can presumably inhibit protein glycation, when associated with the methylglyoxal inevitably produced during glycolysis. Herein, we hypothesized a nonenzymatic interaction between putrescine and methylglyoxal in putrescine-deficient or -overexpressing Dictyostelium cells in high-glucose medium, which can control methylglyoxal production. Putrescine was essentially required for growth rescue accompanying methylglyoxal detoxification when cells underwent growth defect and cell cycle G1-arrest when supplemented with high glucose. Furthermore, methylglyoxal regulation by putrescine seemed to be a parallel pathway independent of the changes in cellular glutathione content in high-glucose medium. Consequently, we suggest that Dictyostelium cells need polyamines for normal growth and cellular methylglyoxal regulation.

Candida albicans erythroascorbate peroxidase regulates intracellular methylglyoxal and reactive oxygen species independently of D-erythroascorbic acid.

Candida albicans D-erythroascorbate peroxidase (EAPX1), which can catalyze the oxidation of D-erythroascorbic acid (EASC) to water, was observed to be inducible in EAPX1-deficient and EAPX1-overexpressing cells via activity staining. EAPX1-deficient cells have remarkably increased intracellular reactive oxygen species and methylglyoxal independent of the intracellular EASC content. The increased methylglyoxal caused EAPX1-deficient cells to activate catalase-peroxidase and cytochrome c peroxidase, which led to defects in cell growth, viability, mitochondrial respiration, filamentation and virulence. These findings indicate that EAPX1 mediates cell differentiation and virulence by regulating intracellular methylglyoxal along with oxidative stresses, regardless of endogenous EASC biosynthesis or alternative oxidase expression.

NAD(+)-linked alcohol dehydrogenase 1 regulates methylglyoxal concentration in Candida albicans.

We purified a fraction that showed NAD(+)-linked methylglyoxal dehydrogenase activity, directly catalyzing methylglyoxal oxidation to pyruvate, which was significantly increased in glutathione-depleted Candida albicans. It also showed NADH-linked methylglyoxal-reducing activity. The fraction was identified as a NAD(+)-linked alcohol dehydrogenase (ADH1) through mass spectrometric analyses. In ADH1-disruptants of both the wild type and glutathione-depleted cells, the intracellular methylglyoxal concentration increased significantly; defects in growth, differentiation, and virulence were observed; and G2-phase arrest was induced.

Glutathione initiates the development of Dictyostelium discoideum through the regulation of YakA.

Reduced glutathione (GSH) is an essential metabolite that performs multiple indispensable roles during the development of Dictyostelium. We show here that disruption of the gene (gcsA-) encoding y-glutamylcysteine synthetase, an essential enzyme in GSH biosynthesis, inhibited aggregation, and that this developmental defect was rescued by exogenous GSH, but not by other thiols or antioxidants. In GSH-depleted gcsA- cells, the expression ofa growth-stage-specific gene (cprD) was not inhibited, and we did not detect the expression of genes that encode proteins required for early development (cAMP receptor, carA/cAR1; adenylyl cyclase, acaA/ACA; and the catalytic subunit of protein kinase A, pkaC/PKA-C). The defects in gcsA cells were not restored by cAMP stimulation or by cAR1 expression. Further, the expression of yakA, which initiates development and induces the expression of PKA-C, ACA, and cAR1, was regulated by the intracellular concentration of GSH. Constitutive expression of YakA in gcsA- cells (YakA(OE)/gcsA-) rescued the defects in developmental initiation and the expression of early developmental genes in the absence of GSH. Taken together, these findings suggest that GSH plays an essential role in the transition from growth to development by modulating the expression of the genes encoding YakA as well as components thatact downstream in the YakA signaling pathway.

Methylglyoxal accumulation by glutathione depletion leads to cell cycle arrest in Dictyostelium.

Reduced glutathione (GSH) serves as a primary redox buffer and its depletion causes growth inhibition or apoptosis in many organisms. In Dictyostelium discoideum, the null mutant (gcsA(-)) of gcsA encoding gamma-glutamylcysteine synthetase shows growth arrest and developmental defect when GSH is depleted. To investigate the mechanism by which GSH depletion induces growth arrest, a proteomic analysis was performed and aldose reductase (AlrA) was identified as the most prominently induced protein in gcsA(-) cells. Induction of AlrA was dependent on GSH concentration and was repressed by GSH but not effectively by either the reducing agent such as dithiothreitol or overexpression of superoxide dismutase. Methylglyoxal (MG), a toxic alpha-ketoaldehyde, strongly induced alrA expression and AlrA catalysed MG reduction efficiently. The alrA knockdown gcsA(-) cells (gcsA(-)/alrA(as)) exhibited more decreased growth rate than gcsA(-) cells, whereas the gcsA(-) cells overexpressing alrA (gcsA(-)/alrA(oe)) showed the recovery of growth rate. Interestingly, intracellular MG levels were significantly augmented in gcsA(-)/alrA(as) cells compared with gcsA(-) cells following GSH depletion. By contrast, gcsA(-)/alrA(oe) cells showed repression of MG induction. Furthermore, MG treatment inhibited growth of wild-type KAx3 cells, inducing G1 phase arrest. Thus, our findings suggest that MG accumulated by GSH depletion inhibits cell growth in Dictyostelium.

Crystal structure of filamentous aggregates of human DJ-1 formed in an inorganic phosphate-dependent manner.

Mutations in the DJ-1 gene have been implicated in the autosomal recessive early onset parkinsonism. DJ-1 is a soluble dimeric protein with critical roles in response to oxidative stress and in neuronal maintenance. However, several lines of evidence suggest the existence of a nonfunctional aggregated form of DJ-1 in the brain of patients with some neurodegenerative diseases. Here, we show that inorganic phosphate, an important anion that exhibits elevated levels in patients with Parkinson disease, transforms DJ-1 into filamentous aggregates. According to the 2.4-A crystal structure, DJ-1 dimers are linearly stacked through P(i)-mediated interactions to form protofilaments, which are then bundled into a filamentous assembly.

Thioredoxin reductase is required for growth and regulates entry into culmination of Dictyostelium discoideum.

The thioredoxin system, consisting of thioredoxin, thioredoxin reductase and NADPH, has been well established to be critical for the redox regulation of protein function and signalling. To investigate the role of thioredoxin reductase (Trr) in Dictyostelium discoideum, we generated mutant cells that underexpress or overexpress Trr. Trr-underexpressing cells exhibited severe defects in axenic growth and development. Trr-overexpressing (TrrOE) cells formed very tiny plaques on a bacterial lawn and had a lower rate of bacterial uptake. When developed in the dark, TrrOE cells exhibited a slugger phenotype, defined by a prolonged migrating slug stage. Like other slugger mutants, they were hypersensitive to ammonia, which has been known to inhibit culmination by raising the pH of intracellular acidic compartments. Interestingly, TrrOE cells showed defective acidification of intracellular compartments and decreased activity of vacuolar H+-ATPase which functions in the acidification of intracellular compartments. Moreover, biochemical studies revealed that the thioredoxin system can directly reduce the catalytic subunit of vacuolar H+-ATPase whose activity is regulated by reversible disulphide bond formation. Taken together, these results suggest that Dictyostelium Trr may be essential for growth and play a role in regulation of phagocytosis and culmination, possibly through the modulation of vacuolar H+-ATPase activity.

Reduced glutathione levels affect the culmination and cell fate decision in Dictyostelium discoideum.

Glutaredoxins have been known to be glutathione-dependent oxidoreductases that participate in the redox regulation of various cellular processes. To understand the role of glutaredoxins in the development, we examined glutaredoxin 1 (Grx1) of Dictyostelium discoideum. Its mRNA was highly accumulated at the mound and the culmination stages. When Grx1-overexpressing cells were developed, their culmination was delayed, and the expression of marker genes for prespore and spore decreased. Interestingly, they had about 1.5-fold higher amount of reduced glutathione (GSH) compared with parental cells and their prolonged migration was repressed by the oxidant such as hydrogen peroxide. To confirm the effect of GSH on the culmination, glutathione reductase (Gsr) was overexpressed or underexpressed. Similar to Grx1-overexpressing cells, Gsr-overexpressing cells contained about 1.5-fold higher amount of GSH and exhibited the delayed culmination. In contrast, the knockdown mutant of Gsr had nearly 50% lower amount of GSH and showed accelerated culmination. Taken together, these data suggest that the culmination of Dictyostelium is controlled by GSH. In addition, the cells having higher GSH levels showed a prestalk tendency in the chimeric slugs with parental cells, indicating that the difference in the amount of GSH may affect the determination of cell fate.

Glutathione is required for growth and prespore cell differentiation in Dictyostelium.

Glutathione (GSH) is the most abundant non-protein thiol in eukaryotic cells and acts as reducing equivalent in many cellular processes. We investigated the role of glutathione in Dictyostelium development by disruption of gamma-glutamylcysteine synthetase (GCS), an essential enzyme in glutathione biosynthesis. GCS-null strain showed glutathione auxotrophy and could not grow in medium containing other thiol compounds. The developmental progress of GCS-null strain was determined by GSH concentration contained in preincubated media before development. GCS-null strain preincubated with 0.2 mM GSH was arrested at mound stage or formed bent stalk-like structure during development. GCS-null strain preincubated with more than 0.5 mM GSH formed fruiting body with spores, but spore viability was significantly reduced. In GCS-null strain precultured with 0.2 mM GSH, prestalk-specific gene expression was delayed, while prespore-specific gene and spore-specific gene expressions were not detected. In addition, GCS-null strain precultured with 0.2 mM GSH showed prestalk tendency and extended G1 phase of cell cycle. Since G1 phase cells at starvation differentiate into prestalk cells, developmental defect of GCS-null strain precultured with 0.2 mM GSH may result from altered cell cycle. These results suggest that glutathione itself is essential for growth and differentiation to prespore in Dictyostelium.

Crystal structure of nickel-containing superoxide dismutase reveals another type of active site.

Superoxide dismutases (SODs, EC 1.15.1.1) are ubiquitous enzymes that efficiently catalyze the dismutation of superoxide radical anions to protect biological molecules from oxidative damage. The crystal structure of nickel-containing SOD (NiSOD) from Streptomyces seoulensis was determined for the resting, x-ray-reduced, and thiosulfate-reduced enzyme state. NiSOD is a homohexamer consisting of four-helix-bundle subunits. The catalytic center resides in the N-terminal active-site loop, where a Ni(III) ion is coordinated by the amino group of His-1, the amide group of Cys-2, two thiolate groups of Cys-2 and Cys-6, and the imidazolate of His-1 as axial ligand that is lost in the chemically reduced state as well as after x-ray-induced reduction. This structure represents a third class of SODs concerning the catalytic metal species, subunit structure, and oligomeric organization. It adds a member to the small number of Ni-metalloenzymes and contributes with its Ni(III) active site to the general understanding of Ni-related biochemistry. NiSOD is shown to occur also in bacteria other than Streptomyces and is predicted to be present in some cyanobacteria.