60 research outputs found
Expression of Candida albicans glutathione transferases is induced inside phagocytes and upon diverse environmental stresses
Candida albicans has four ORFs for glutathione transferases (GSTs) of the GTT
classes, and another one coding for an Omega class member. Under laboratory
conditions, only GTT11 (GTT1/2 class) and GTO1 (Omega class) are expressed
significantly in exponentially growing cells, particularly when these are subjected
to diverse environmental stresses, including oxidative stress. They also become
transitorily upregulated at the early stationary phase. Accordingly, the levels of the
CaGto1 and CaGtt11 proteins increase after treatment with oxidants and upon
osmotic stress, in addition to the early stationary phase. GTT11 and GTO1
transcription shows a complex dependence on the Hog1 and Cap1 factors upon
different stresses. Purified CaGtt11 and CaGto1 proteins display enzyme activities
similar to the Saccharomyces cerevisiae homologues. Thus, CaGtt11 has activity
against standard GST substrates and is also active as peroxidase, while CaGto1
displays thiol oxidoreductase and dehydroascorbate reductase activities. Fluores-
cence microscopy and subfractionation studies indicate that CaGto1 is cytosolic,
while CaGtt11 is associated with a particulate fraction. Under ex vivo conditions, CaGto1 and CaGtt11 become transitorily upregulated inside macrophages and
neutrophils. Under these conditions, the promoter of GTT14 (GTT1/2 class) also
becomes activated. These observations point to the importance of C. albicans
GSTs in the defence against phagocytes
Selenite-induced cell death in Saccharomyces cerevisiae: protective role of glutaredoxins
Unlike in higher organisms, selenium is not essential for growth in Saccharomyces cerevisiae. In this species, it causes toxic effects at high concentrations. In the present study, we show that when supplied as selenite to yeast cultures growing under fermentative metabolism, its effects can be dissected into two death phases. From the time of initial treatment, it causes loss of membrane integrity and genotoxicity. Both effects occur at higher levels in mutants lacking Grx1p and Grx2p than in wild-type cells, and are reversed by expression of a cytosolic version of the membrane-associated Grx7p glutaredoxin. Grx7p can also rescue the high levels of protein carbonylation damage that occur in selenite-treated cultures of the grx1 grx2 mutant. After longer incubation times, selenite causes abnormal nuclear morphology and the appearance of TUNEL-positive cells, which are considered apoptotic markers in yeast cells. This effect is independent of Grx1p and Grx2p. Therefore, the protective role of the two glutaredoxins is restricted to the initial stages of selenite treatment. Lack of Yca1p metacaspase or of a functional mitochondrial electron transport chain only moderately diminishes apoptotic-like death by selenite. In contrast, selenite-induced apoptosis is dependent on the apoptosis-inducing factor Aif1p. In the absence of the latter, intracellular protein carbonylation is reduced after prolonged selenite treatment, supporting the supposition that part of the oxidative damage is contributed by apoptotic cells
Monothiol glutaredoxins: a common domain for multiple functions
Monothiol glutaredoxins with the CGFS sequence at the active site are widespread among prokaryotes and eukaryotes. Two subclasses exist, those with a single glutaredoxin domain and those with a thioredoxin-like region followed by one or
more glutaredoxin domains. Studies in Saccharomyces cerevisiae have demonstrated the role of the Grx5 protein in the biogenesis of iron-sulphur clusters. Grx5 homologues in other eukaryotes could carry out similar functions. Two S. cerevisiae monothiol glutaredoxins with the thioredoxin-like extension, Grx3 and Grx4, are modulators of the transcriptional activator Aft1, which regulates iron uptake in yeast. The human PICOT protein is a Grx3/Grx4 homologue with the same hybrid primary structure, which regulates protein kinase C activity and may participate in physiological processes such as control of cardiac function. Therefore, monothiol glutaredoxins share a common basic structural motif and biochemical mechanism of action, while participating in a diversity of cellular functions as protein redox regulators
Altered intracellular calcium homeostasis and endoplasmic reticulum redox state in Saccharomyces cerevisiae cells lacking Grx6 glutaredoxin
Glutaredoxin 6 (Grx6) of Saccharomyces cerevisiae is an integral thiol oxidoreductase protein of the endoplasmic reticulum/Golgi vesicles. Its absence alters the redox equilibrium of the reticulum lumen toward a more oxidized state, thus compensating the defects in protein folding/secretion and cell growth caused by low levels of the oxidase Ero1. In addition, null mutants in GRX6 display a more intense unfolded protein response than wild-type cells upon treatment with inducers of this pathway. These observations support a role of Grx6 in regulating the glutathionylation of thiols of endoplasmic reticulum/Golgi target proteins and consequently the equilibrium between reduced and oxidized glutathione in the lumen of these compartments. A specific function influenced by Grx6 activity is the homeostasis of intracellular calcium. Grx6-deficient mutants have reduced levels of calcium in the ER lumen, whereas accumulation occurs at the cytosol from extracellular sources. This results in permanent activation of the calcineurin-dependent pathway in these cells. Some but not all the phenotypes of the mutant are coincident with those of mutants deficient in intracellular calcium transporters, such as the Golgi Pmr1 protein. The results presented in this study provide evidence for redox regulation of calcium homeostasis in yeast cells.This work was supported by Grants BFU2010-17656 (Ministerio de Economía y Competitividad, Spain) and 2009/SGR/196 (Generalitat de Catalunya)
Structural and functional diversity of glutaredoxins in yeast
Glutaredoxins are defined as thiol disulfide oxidoreductases that reduce disulfide bonds employing reduced glutathione as electron donor. They constitute a
complex family of proteins with a diversity of enzymatic and functional properties.
Thus, dithiol glutaredoxins are able to reduce disulfide bonds and deglutathionylate
mixed disulfides between glutathione and cysteine protein residues. They could act
regulating the redox state of sulfhydryl residues of specific proteins, while thioredoxins (another family of thiol disulfide oxidoreductases which employ NADPH as electron donor) would be the general sulfhydryl reductants. Some dithiol glutaredoxins such as human Grx2 form dimers bridged by one iron-sulfur cluster, which acts as a sensor of oxidative stress, therefore regulating the activity of the glutaredoxin. The ability to interact with iron-sulfur clusters as ligands is also characteristic of monothiol glutaredoxins with a CGFS-type active site. These do not display thiol oxidoreductase activity, but have roles in iron homeostasis. The three members of this subfamily in Saccharomyces cerevisiae participate in the synthesis of the iron-sulfur clusters in mitochondria (Grx5), or in signalling the iron status inside the cell for regulation of iron uptake and intracellular iron relocalization (Grx3 and Grx4). Such role in iron metabolism seems to be evolutionary conserved. Fungal cells also contain membraneassociated
glutaredoxins structurally and enzymatically similar to dithiol glutaredoxins,
which may act as redox regulators at the early stages of the protein secretory machinery
Comparative genomics of yeast species: new insights into their biology
The genomes of two hemiascomycetous yeasts
(Saccharomyces cerevisiae and Candida albicans) and
one archiascomycete (Schizosaccharomyces pombe) have
been completely sequenced and the genes have been
annotated. In addition, the genomes of 13 more Hemiascomycetes
have been partially sequenced. The amount
of data thus obtained provides information on the evolutionary
relationships between yeast species. In addition,
the differential genetic characteristics of the
microorganisms explain a number of distinctive biological
traits. Gene order conservation is observed between
phylogenetically close species and is lost in distantly
related species, probably due to rearrangements of short
regions of DNA. However, gene function is much more
conserved along evolution. Compared to S. cerevisiae
and S. pombe, C. albicans has a larger number of specific
genes, i.e., genes not found in other organisms, a fact
that can account for the biological characteristics of this
pathogenic dimorphic yeast which is able to colonize a
large variety of environments
Isolation and characterization of Saccharomyces cerevisiae mutants resistant to aculeacin A
Aculeacin A is a lipopeptide that inhibits ,B-glucan synthesis in yeasts. A number of Saccharomyces cerevisiae mutants resistant to this antibiotic were isolated, and four loci (ACRI, ACR2, ACR3, and ACR4) whose products are involved in the sensitivity to aculeacin A of yeast ceils were defined. Mutants containing mutations
in the four loci were also resistant to echinocandin B, another member of this lipopeptide family of antibiotics. In contrast, acri, acr3, and acr4 mutants were resistant to papulacandin B (an antibiotic containing a disaccharide linked to two fatty acid chains that also inhibits P-glucan synthesis), but acr2 mutants were
susceptible'to this antibiotic. This result defines common and specific steps in the entry and action of aculeacin A and papulacandin B. The analysis of double mutants revealed an epistatic effect of the acr2 mutation on the other three mutations. Cell walls of the four different mutants did not show significant alterations in composition with respect to the parental strain, and in vitro glucan synthase activity was also unaffected. However, cell surface hydrophobicity in three of the mutants was considerably decreased with respect to the parental strain
Impaired mitochondrial Fe-S cluster biogenesis activates the DNA damage response through different signaling mediators
Fe-S cluster biogenesis machinery is required for multiple DNA
metabolism processes. In this work, we show that, in Saccharomyces
cerevisiae, defects at different stages of the mitochondrial Fe-S
cluster assembly machinery (ISC) result in increased spontaneous
mutation rate and hyper-recombination, accompanied by an
increment in Rad52-associated DNA repair foci and a higher
phosphorylated state of γH2A histone, altogether supporting the
presence of constitutive DNA lesions. Furthermore, ISC assembly
machinery deficiency elicits a DNA damage response that
upregulates ribonucleotide reductase activity by promoting the
reduction of Sml1 levels and the cytosolic redistribution of Rnr2 and
Rnr4 enzyme subunits. Depending on the impaired stage of the ISC
machinery, different signaling pathway mediators contribute to such a
response, converging on Dun1. Thus, cells lacking the glutaredoxin
Grx5, which are compromised at the core ISC system, show Mec1-
and Rad53-independent Dun1 activation, whereas both Mec1 and
Chk1 are required when the non-core ISC member Iba57 is absent.
Grx5-null cells exhibit a strong dependence on the error-free postreplication
repair and the homologous recombination pathways,
demonstrating that a DNA damage response needs to be activated
upon ISC impairment to preserve cell viability.This work was supported by the Ministerio de Economia y Competitividad (MINECO, ́
Spain) [grants numbers BFU2010-17656 and CSD2007-0020]. J.P. was the
recipient of a predoctoral grant from MINEC
Glutaredoxins in fungi
Glutaredoxins (GRXs) can be subdivided into two subfamilies: dithiol GRXs with the
CPY/FC active site motif, and monothiol GRXs with the CGFS motif. Both subfamilies share a thioredoxin-fold structure. Monothiol GRXs exist with a single Grx domain while others have a thioredoxin-like domain (Trx) and one or more Grx domains in tandem. Most fungi have both dithiol and monothiol GRXs with different subcellular locations. GRX-like molecules also exist in fungi that separate in one residue from one of the canonical active site motifs. Additionally, Omega-class glutathione transferases are active as GRXs. Among fungi, the GRXs more extensively studied are those from Saccharomyces cerevisiae. This organism contains two dithiol GRXs (ScGrx1 and ScGrx2) with partially overlapping functions in defence against oxidative stress. In this function, they cooperate with glutathione transferases Gtt1 and Gtt2. While ScGrx1 is cytosolic, two pools exist for ScGrx2, a major one at the cytosol and a minor one at mitocondria. On the other hand, S. cerevisiae cells have two monothiol GRXs with the Trx-Grx structure (ScGrx3 and ScGrx4) that locate at the nucleus and probably regulate the activity of transcription factors such as Aft1, and one monothiol glutaredoxin with the Grx structure (ScGrx5) that localizes as the mitochondria matrix, where it participates in the synthesis of iron-sulfur clusters. The function of yeast Grx5 seems to be conserved along the evolutionary scale
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