Articles publicats (IRBLleida)

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https://repositori.udl.cat/handle/10459.1/59998

L'IRBLleida és un centre de recerca conjunt entre la Universitat i el Departament de Salut de la Generalitat de Catalunya. Té com a funció potenciar les sinergies de recerca biomèdica entre ambdós institucions i està compromès en avançar en la recerca biomèdica com a mitja per millorar la salut de la població i facilitar una activitat assistencial òptima en situacions de malaltia. [Més informació].

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Analysis of U1snRNP-specific A protein cross-linked complexes
(Wiley, 1990) Boix Torras, Jacint; Habets, W.J; van Venrooij, W.J.; Smith, H.C.
The organization of the U1snRNP-specific A protein (34 kDa) has been analyzed by 12 and 16 A thiol-reversible chemical cross-linking and Western blotting. A-containing cross-linked complexes had molecular masses of 43, 47, 56, 62, 67, 105 and 125 kDa. None of these complexes could be cross-linked following ribonuclease digestion, suggesting that UsnRNA may play important roles in the spatial organization of A and other proteins. Moreover, the data suggest that A is proximal to, and may have interactions with, UsnRNP-specific proteins C and 70 kDa as well as with UsnRNP-common proteins B, E and G.
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Separation of rooster spermatogenic nuclei by means of centrifugal elutriation
(Wiley, 1993) Boix Torras, Jacint; Roca, Joaquim
By application of minor variations in the usual method of centrifugal elutriation, we have been able to separate a nuclear suspension from rooster testes. We have obtained a population of elongated or late spermatid nuclei (98% purity), a Key terms: Centrifugal elutriation, nupopulation of round or early spermatid clei separation, rooster spermatogenesis nuclei (90% purity), and, finally, a mixed population of meiotic and pre-meiotic nuclei.
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Characterization of the Cell Death Process Induced by Staurosporine in Human Neuroblastoma Cell Lines
(Elsevier, 1997) Boix Torras, Jacint; Llecha Cano, Núria; Yuste Mateos, Víctor J. (Víctor José); Comella i Carnicé, Joan Xavier
Staurosporine is a potent and non-specific inhibitor of protein kinases. There is also evidence of staurosporine being a potent inducer of apoptosis. In several human neuroblastoma cell lines (SH-SY5Y, NB69, IMR-5 and IMR-32) we have found 100 nM staurosporine to induce cell death in half the population (EC50). Electron microscopy of these cells, fluorescence microscopy after Hoechst-33258 staining of chromatin and agarose-electrophoresis of DNA, show two different types of cell death. SH-SY5Y and NB69 die by apoptosis and display all the characteristic features of it. IMR-5 and IMR-32 lack some of these features and a ladder pattern of DNA degradation is not found. Different morphological types of apoptosis have been described during the development of vertebrates; the possibility of finding a similar diversity in cell culture is suggested. On the other hand, staurosporine is a potent promoter of neurite outgrowth. In all the neuroblastoma cell lines we have tested, neurite-promoting and cell death-inducing staurosporine concentrations mostly overlap. This fact has not been reported before, probably because of an early versus late timing of these two different phenomena. The neuritogenic effect has prompted the suggestion that staurosporine could be a prototype of drugs for neurodegenerative diseases; the present study raises several concerns about such a proposal.
Open Access
Mitochondrial membrane peroxidizability index is inversely related to maximum life span in mammals
(Elsevier, 1998) Pamplona Gras, Reinald; Portero Otín, Manuel; Riba, David; Ruiz, Cristina; Prat Corominas, Joan; Bellmunt, Maria Josep; Barja, Gustavo
The oxidative stress theory of aging predicts a low degree of fatty acid unsaturation in tissues of longevous animals, because membrane lipids increase their sensitivity to oxidative damage as a function of their unsaturation. Accordingly, the fatty acids analyses of liver mitochondria from eight mammals, ranging in maximum life span from 3.5 to 46 years, show that the total number of double bonds and the peroxidizability index are negatively correlated with maximum life span (r = −0. 88, P < 0.003; r = −0.87, P < 0.004, respectively). This is not due to a low content of unsaturated fatty acids in longevous animals, but mainly to a redistribution between kinds of the polyunsaturated n–3 fatty acids series, shifting from the highly unsaturated docosahexaenoic acid (r = −0.89, P < 0.003) to the less unsaturated linolenic acid (r = 0.97, P < 0.0001). This redistribution pattern strongly suggests the presence of a constitutively low Δ6-desaturase activity in longevous animals (r = −0.96, P < 0.0001). Thus, it may be proposed that, during evolution, a low degree of fatty acid unsaturation in liver mitochondria may have been selected in longevous mammals in order to protect the tissues against oxidative damage, while maintaining an appropriate environment for membrane function.
Open Access
Neonatal tumor necrosis factor alpha promotes diabetes in nonobese diabetic mice by CD154-independent antigen presentation to CD8(+) T cells
(Rockefeller University Press, 2000) Green, E. Allison; Wong, F. Susan; Eshima, Koji; Mora Giral, Concepció; Flavell, Richard A.
Neonatal islet-specific expression of tumor necrosis factor (TNF)-α in nonobese diabetic mice promotes diabetes by provoking islet-infiltrating antigen-presenting cells to present islet peptides to autoreactive T cells. Here we show that TNF-α promotes autoaggression of both effector CD4+ and CD8+ T cells. Whereas CD8+ T cells are critical for diabetes progression, CD4+ T cells play a lesser role. TNF-α–mediated diabetes development was not dependent on CD154–CD40 signals or activated CD4+ T cells. Instead, it appears that TNF-α can promote cross-presentation of islet antigen to CD8+ T cells using a unique CD40–CD154-independent pathway. These data provide new insights into the mechanisms by which inflammatory stimuli can bypass CD154–CD40 immune regulatory signals and cause activation of autoreactive T cells.
Open Access
Oxidative stress promotes specific protein damage in Saccharomyces cerevisiae
(The American Society for Biochemistry and Molecular Biology, 2000) Cabiscol Català, Elisa; Piulats Combalia, Eva; Echave Lozano, Pedro; Herrero Perpiñán, Enrique; Ros Salvador, Joaquim
We have analyzed the proteins that are oxidatively damaged when Saccharomyces cerevisiae cells are ex- posed to stressing conditions. Carbonyl groups generated by hydrogen peroxide or menadione on proteins of aero- bically respiring cells were detected by Western blotting, purified, and identified. Mitochondrial proteins such as E2 subunits of both pyruvate dehydrogenase and ␣-keto- glutarate dehydrogenase, aconitase, heat-shock protein 60, and the cytosolic fatty acid synthase (␣ subunit) and glyceraldehyde-3-phosphate dehydrogenase were the ma- jor targets. In addition we also report the in vivo modifi- cation of lipoamide present in the above-mentioned E2 subunits under the stressing conditions tested and that this also occurs with the homologous enzymes present in Escherichia coli cells that were used for comparative analysis. Under fermentative conditions, the main pro- tein targets in S. cerevisiae cells treated with hydrogen peroxide or menadione were pyruvate decarboxylase, enolase, fatty acid synthase, and glyceraldehyde-3-phos- phate dehydrogenase. Under the stress conditions tested, fermenting cells exhibit a lower viability than aerobically respiring cells and, consistently, increased peroxide gen- eration as well as higher content of protein carbonyls and lipid peroxides. Our results strongly suggest that the oxidative stress in prokaryotic and eukaryotic cells shares common features.
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Osmotic stress causes a G1 cell cycle delay and downregulation of Cln3/Cdc28 activity in Saccharomyces cerevisiae
(Blackwell Science, 2001) Bellí i Martínez, Gemma; Garí Marsol, Eloi; Aldea, Martí; Herrero Perpiñán, Enrique
Moderate hyperosmotic stress on Saccharomyces cerevisiae cells produces a temporary delay at the G1 stage of the cell cycle. This is accompanied by transitory downregulation of CLN1, CLN2 and CLB5 transcript levels, although not of CLN3, which codes for the most upstream activator of the G1/S transition. Osmotic shock to cells synchronized in early G1, when Cln3 is the only cyclin present, causes a delay in cell cycle resumption. This points to Cln3 as being a key cell cycle target for osmotic stress. We have observed that osmotic shock causes downregulation of the kinase activity of Cln3±Cdc28 complexes. This is concomitant with a temporary accumulation of Cln3 protein as a result of increased stability. The effects of the osmotic stress in G1 are not suppressed in CLN3-1 cells with increased kinase activity, as the Cln3±Cdc28 activity in this mutant is still affected by the shock. Although Hog1 is not required for the observed cell cycle arrest in hyperosmotic conditions, it is necessary to resume the cell cycle at KCl concentrations higher than 0.4 M.
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Isolation of a Candida albicans gene, tightly linked to URA3, coding for a putative transcription factor that suppresses a Saccharomyces cerevisiae aft1 mutation
(John Wiley & Sons, 2001) Gómez García, Micaela; O'Connor, José-Enrique; Latorre García, Lorena; Irar Martínez, Sami; Herrero Perpiñán, Enrique; Del Castillo Agudo, Lucas
A pathogen such as C. albicans needs an ef®cient mechanism of iron uptake in an iron- restricted environment such as is the human body. A ferric-reductase activity regulated by iron and copper, and analogous to that in S. cerevisiae, has been described in C. albicans. We have developed an in-plate protocol for the isolation of clones that complement an aft1 mutation in S. cerevisiae that makes cells dependent on iron for growth. After transformation of S. cerevisiae aft1 with a C. albicans library, we have selected clones that grow in conditions of iron de®ciency and share an identical plasmid, pIRO1, with a 4500 bp insert containing the URA3 gene and an ORF (IRO1) responsible for the suppression of the iron dependency. IRO1 does not show homology with AFT1 or with other sequences in the databases. Northern analysis demonstrates constitutive expression of IRO1. CAI4, a C. albicans strain isolated as Dura3, also has a deletion of the 3k half of IRO1, and displays in YNB medium similar phenotypic characteristics to S. cerevisiae aft1 mutant strains. Therefore, we consider IRO1 as a gene of C. albicans involved in the utilization of iron. However, in extreme conditions of iron deprivation, CAI4 seems to activate alternative mechanisms of iron uptake that allow a better growth than the wild strain SC5314. Analysis of its predicted protein sequence is in agreement with a role of Iro1p as a transcription factor.
Open Access
Sit4 is required for proper modulation of the biological functions mediated by Pkc1 and the cell integrity pathway in Saccharomyces cerevisiae
(The American Society for Biochemistry and Molecular Biology, 2002) Torre Ruiz, M. A. de la; Torres Rosell, Jordi; Ariño Carmona, Joaquín; Herrero Perpiñán, Enrique
Maintenance of cellular integrity in Saccharomyces cerevisiae is carried out by the activation of the protein kinase C-mediated mitogen-activated protein kinase (PKC1-MAPK) pathway. Here we report that correct down-regulation of both basal and induced activity of the PKC1-MAPK pathway requires the SIT4 function. Sit4 is a protein phosphatase also required for a proper cell cycle progression. We present evidence demonstrat- ing that the G1 to S delay in the cell cycle, which occurs as a consequence of the absence of Sit4, is mediated by up-regulation of Pkc1 activity. Sit4 operates down- stream of the plasma membrane sensors Mid2, Wsc1, and Wsc2 and upstream of Pkc1. Sit4 affects all known bio- logical functions involving Pkc1, namely Mpk1 activity and cell wall integrity, actin cytoskeleton organization, and ribosomal gene transcription.
Open Access
Structure-function analysis of yeast Grx5 monothiol glutaredoxin defines essential amino acids for the function of the protein
(The American Society for Biochemistry and Molecular Biology, 2002) Bellí i Martínez, Gemma; Polaina, Julio; Tamarit Sumalla, Jordi; Torre Ruiz, M. A. de la; Rodríguez Manzanaque, Maria Teresa; Ros Salvador, Joaquim; Herrero Perpiñán, Enrique
Grx5 defines a family of yeast monothiol glutaredox- ins that also includes Grx3 and Grx4. All three proteins display significant sequence homology with proteins found from bacteria to humans. Grx5 is involved in iron/ sulfur cluster assembly at the mitochondria, but the function of Grx3 and Grx4 is unknown. Three-dimen- sional modeling based on known dithiol glutaredoxin structures predicted a thioredoxin fold structure for Grx5. Positionally conserved amino acids in this glu- taredoxin family were replaced in Grx5, and the effect on the biological function of the protein has been tested. For all changes studied, there was a correlation between the effects on several different phenotypes: sensitivity to oxidants, constitutive protein oxidation, ability for respiratory growth, auxotrophy for a number of amino acids, and iron accumulation. Cys60 and Gly61 are essen- tial for Grx5 function, whereas other single or double substitutions in the same region had no phenotypic ef- fects. Gly115 and Gly116 could be important for the for- mation of a glutathione cleft on the Grx5 surface, in contrast to adjacent Cys117. Substitution of Phe50 alters the ␤-sheet in the thioredoxin fold structure and inhib- its Grx5 function. None of the substitutions tested affect the structure at a significant enough level to reduce protein stability.
Open Access
Regulation of the cell integrity pathway by rapamycin-sensitive TOR function in budding yeast
(The American Society for Biochemistry and Molecular Biology, 2002) Torres Rosell, Jordi; Di Como, Charles J.; Herrero Perpiñán, Enrique; Torre Ruiz, M. A. de la
The TOR (target of rapamycin) pathway controls cell growth in response to nutrient availability in eukary- otic cells. Inactivation of TOR function by rapamycin or nutrient exhaustion is accompanied by triggering various cellular mechanisms aimed at overcoming the nutrient stress. Here we report that in Saccharomyces cerevisiae the protein kinase C (PKC)-mediated mito- gen-activated protein kinase pathway is regulated by TOR function because upon specific Tor1 and Tor2 in- hibition by rapamycin, Mpk1 is activated rapidly in a process mediated by Sit4 and Tap42. Osmotic stabiliza- tion of the plasma membrane prevents both Mpk1 acti- vation by rapamycin and the growth defect that occurs upon the simultaneous absence of Tor1 and Mpk1 func- tion, suggesting that, at least partially, TOR inhibition is sensed by the PKC pathway at the cell envelope. This process involves activation of cell surface sensors, Rom2, and downstream elements of the mitogen- activated protein kinase cascade. Rapamycin also in- duces depolarization of the actin cytoskeleton through the TOR proteins, Sit4 and Tap42, in an osmotically suppressible manner. Finally, we show that entry into stationary phase, a physiological situation of nutrient depletion, also leads to the activation of the PKC path- way, and we provide further evidence demonstrating that Mpk1 is essential for viability once cells enter G0.
Open Access
Mitochondrial Hsp60, resistance to oxidative stress, and the labile iron pool are closely connected in Saccharomyces cerevisiae
(The American Society for Biochemistry and Molecular Biology, 2002) Cabiscol Català, Elisa; Bellí i Martínez, Gemma; Tamarit Sumalla, Jordi; Echave Lozano, Pedro; Herrero Perpiñán, Enrique; Ros Salvador, Joaquim
In the present study, we have analyzed the role of the molecular chaperone Hsp60 in protection of Saccharo- myces cerevisiae against oxidative damage. We con- structed mutant strains in which the levels of Hsp60 protein, compared with wild-type cells, were four times greater, and the addition of doxycycline gradually re- duces them to 20% of wild-type. Under oxidative-stress conditions, the progressive decrease in Hsp60 levels in these mutants resulted in reduced cell viability and an increase in both cell peroxide species and protein car- bonyl content. Protection of Fe/S-containing enzymes from oxidative inactivation was found to be dose-de- pendent with respect to Hsp60 levels. As these enzymes release their iron ions under oxidative-stress condi- tions, the intracellular labile iron pool, monitored with calcein, was higher in cells with reduced Hsp60 levels. Consistently, the iron chelator deferoxamine protected low Hsp60-expressing cells from both oxidant-induced death and protein oxidation. These results indicate that the role of Hsp60 in oxidative-stress defense is explained by protection of several Fe/S proteins, which prevent the release of iron ions and thereby avert further damage.
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Characterization of a Candida albicans gene encoding a putative transcriptional factor required for cell wall integrity
(Elsevier, 2003) Moreno, Inmaculada; Pedreño, Yolanda; Maicas i Prieto, Sergio; Sentandreu, Rafael; Herrero Perpiñán, Enrique; Valentín, Eulogio
After screening a Candida albicans genome database the product of an open reading frame (ORF) (CA2880) with 49% homology to the product of Saccharomyces cerevisiae YPL133c, a putative transcriptional factor, was identified. The disruption of the C. albicans gene leads to a major sensitivity to calcofluor white and Congo red, a minor sensitivity to sodium dodecyl sulfate, a major resistance to zymolyase, and an alteration of the chemical composition of the cell wall. For these reasons we called it CaCWT1 (for C. albicans cell wall transcription factor). CaCwt1p contains a putative Zn(II) Cys(6) DNA binding domain characteristic of some transcriptional factors and a PAS domain. The CaCWT1 gene is more expressed in stationary phase cells than in cells growing exponentially. To our knowledge, this is the first Zn(II) Cys(6) transcriptional factor-encoding gene implicated in the cell wall architecture.
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Prokaryotic and eukaryotic monothiol glutaredoxins are able to perform the functions of Grx5 in the biogenesis of Fe/S clusters in yeast mitochondria
(Elsevier, 2003) Molina Navarro, Maria Micaela; Casas Herranz, Celia; Piedrafita Llorens, Lídia; Bellí i Martínez, Gemma; Herrero Perpiñán, Enrique
The Saccharomyces cerevisiae monothiol glutare- doxin Grx5 participates in the mitochondrial biogenesis of iron–sulfur clusters. Grx5 homologues exist in organisms from bacteria to humans. Chicken (cGRX5) and human (hGRX5) homologues contain a mitochondrial targeting sequence, suggest- ing a mitochondrial localization for these two proteins. We have compartmentalized the Escherichia coli and Synechocystis sp. homologues, and also cGRX5 and hGRX5, in the mitochondrial matrix of a yeast grx5 mutant. All four heterologous proteins rescue the defects of the mutant. The chicken cGRX5 gene was significantly expressed throughout the embryo stages in different tissues. These results underline the functional conservation of Grx5 homologues throughout evolution.
Open Access
Biochemical characterization of yeast mitochondrial Grx5 monothiol glutaredoxin
(The American Society for Biochemistry and Molecular Biology, 2003) Tamarit Sumalla, Jordi; Bellí i Martínez, Gemma; Cabiscol Català, Elisa; Herrero Perpiñán, Enrique; Ros Salvador, Joaquim
Grx5 is a yeast mitochondrial protein involved in iron- sulfur biogenesis that belongs to a recently described family of monothiolic glutaredoxin-like proteins. No member of this family has been biochemically charac- terized previously. Grx5 contains a conserved cysteine residue (Cys-60) and a non-conserved one (Cys-117). In this work, we have purified wild type and mutant C60S and C117S proteins and characterized their biochemical properties. A redox potential of ؊175 mV was calculated for wild type Grx5. The pKa values obtained by titration of mutant proteins with iodoacetamide at different pHs were 5.0 for Cys-60 and 8.2 for Cys-117. When Grx5 was incubated with glutathione disulfide, a transient mixed disulfide was formed between glutathione and the cys- tein 60 of the protein because of its low pKa. Binding of glutathione to Cys-60 promoted a decrease in the Cys- 117 pKa value that triggered the formation of a disulfide bond between both cysteine residues of the protein, in- dicating that Cys-117 plays an essential role in the cat- alytic mechanism of Grx5. The disulfide bond in Grx5 could be reduced by GSH but at a rate at least 20 times slower than that observed for the reduction of glutare- doxin 1 from E. coli, a dithiolic glutaredoxin. This slow reduction rate could suggest that GSH may not be the physiologic reducing agent of Grx5. The fact that wild type Grx5 efficiently reduced a glutathiolated protein used as a substrate indicated that Grx5 may act as a thiol reductase inside the mitochondria.
Open Access
Comparative genomics of yeast species: new insights into their biology
(Sociedad Española de Microbiología, 2003) Herrero Perpiñán, Enrique; Torre Ruiz, M. A. de la; Valentín, Eulogio
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.
Open Access
Relationships between big five personality factors and values
(Society for Personality Research, 2004) Aluja Fabregat, Antón; García Rodríguez, Luis Francisco
An analysis of principal components showed a robust structure of three factors formed by 25 of the 30 values studied. These factors were called Social Power, Order and Benevolence values. Those factors match with some of Schwartz’s (1992) types of values. Sex differences and correlations between traits and values replicate other authors’ findings. Path analysis results, performed separately for females and males, yielded a better fit for the female than for the male group. However, although there were no substantial differences with regard to the prediction of Surgency, Conscientiousness, Unfriendliness and Neuroticism, Order values (order, neatness, responsibility) for males and Social Power values (power, prestige, fame) for females predicted the Intellect trait. This pattern of results is discussed in relation to changes in gender roles.
Open Access
Saccharomyces cerevisiae glutaredoxin 5-deficient cells subjected to continuous oxidizing conditions are affected in the expression of specific sets of genes
(The American Society for Biochemistry and Molecular Biology, 2004) Bellí i Martínez, Gemma; Molina Navarro, Maria Micaela; García-Martínez, José; Pérez-Ortín, José E.; Herrero Perpiñán, Enrique
The Saccharomyces cerevisiae GRX5 gene codes for a mitochondrial glutaredoxin involved in the synthesis of iron/sulfur clusters. Its absence prevents respiratory growth and causes the accumulation of iron inside cells and constitutive oxidation of proteins. Null ⌬grx5 mu- tants were used as an example of continuously oxidized cells, as opposed to situations in which oxidative stress is instantaneously caused by addition of external oxi- dants. Whole transcriptome analysis was carried out in the mutant cells. The set of genes whose expression was affected by the absence of Grx5 does not significantly overlap with the set of genes affected in respiratory petite mutants. Many Aft1-dependent genes involved in iron utilization that are up-regulated in a frataxin mu- tant were also up-regulated in the absence of Grx5. BIO5 is another Aft1-dependent gene induced both upon iron deprivation and in ⌬grx5 cells; this links iron and biotin metabolism. Other genes are specifically affected under the oxidative conditions generated by the grx5 muta- tion. One of these is MLP1, which codes for a homologue of the Slt2 kinase. Cells lacking MLP1 and GRX5 are hypersensitive to oxidative stress caused by external agents and exhibit increased protein oxidation in rela- tion to single mutants. This in turn points to a role for Mlp1 in protection against oxidative stress. The genes of the Hap4 regulon, which are involved in respiratory metabolism, are down-regulated in ⌬grx5 cells. This ef- fect is suppressed by HAP4 overexpression. Inhibition of respiratory metabolism during continuous moderately oxidative conditions could be a protective response by the cell.
Open Access
Evolution and cellular function of monothiol glutaredoxins: involvement in iron-sulphur cluster assembly
(Wiley Interscience, 2004) Vilella Mitjana, Felipe; Alves, Rui; Rodríguez-Manzaneque Martínez, María Teresa; Bellí i Martínez, Gemma; Swaminathan, Swarna; Sunnerhagen, Per; Herrero Perpiñán, Enrique
A number of bacterial species, mostly proteobacteria, possess monothiol glutaredoxins homologous to the Saccharomyces cerevisiae mitochondrial protein Grx5, which is involved in iron–sulphur cluster synthesis. Phylogenetic profiling is used to predict that bacterial monothiol glutaredoxins also participate in the iron–sulphur cluster (ISC) assembly machinery, because their phylogenetic profiles are similar to the profiles of the bacterial homologues of yeast ISC proteins. High evolutionary cooccurrence is observed between the Grx5 homologues and the homologues of the Yah1 ferredoxin, the scaffold proteins Isa1 and Isa2, the frataxin protein Yfh1 and the Nfu1 protein. This suggests that a specific functional interaction exists between these ISC machinery proteins. Physical interaction analyses using low-definition protein docking predict the formation of strong and specific complexes between Grx5 and several components of the yeast ISC machinery. Two-hybrid analysis has confirmed the in vivo interaction between Grx5 and Isa1. Sequence comparison techniques and cladistics indicate that the other two monothiol glutaredoxins of S. cerevisiae, Grx3 and Grx4, have evolved from the fusion of a thioredoxin gene with a monothiol glutaredoxin gene early in the eukaryotic lineage, leading to differential functional specialization. While bacteria do not contain these chimaeric glutaredoxins, in many eukaryotic species Grx5 and Grx3/4-type monothiol glutaredoxins coexist in the cell.
Open Access
Nuclear monothiol glutaredoxins of Saccharomyces cerevisiae can function as mitochondrial glutaredoxins
(The American Society for Biochemistry and Molecular Biology, 2004) Molina Navarro, Maria Micaela; Bellí i Martínez, Gemma; Torre Ruiz, M. A. de la; Rodríguez Manzanaque, Maria Teresa; Herrero Perpiñán, Enrique
Glutaredoxins are thiol oxidoreductases that regulate protein redox state. In Saccharomyces cerevisiae, Grx1 and Grx2 are cytosolic dithiol glutaredoxins, whereas Grx3, Grx4, and Grx5 are monothiol glutaredoxins. Grx5 locates at the mitochondrial matrix and is needed for iron/sulfur cluster biogenesis. Its absence causes pheno- types such as inactivation of iron/sulfur enzymes and sensitivity to oxidative stress. Whereas Grx5 contains a single glutaredoxin domain, in Grx3 and Grx4 a thio- redoxin-like domain is fused to the glutaredoxin do- main. Here we have shown that Grx3 locates at the nu- cleus and that the thioredoxin-like domain is required for such location. We have addressed the functional di- vergence among glutaredoxins by targeting Grx2/3/4 molecules to the mitochondrial matrix using the Grx5 targeting sequence. The mitochondrial forms of Grx3 and Grx4 partially rescue the defects of a grx5 null mu- tant. On the contrary, mitochondrially targeted Grx2 does not suppress the mutant phenotype. Both the thi- oredoxin-like and glutaredoxin domains are needed for the mitochondrial activity of Grx3, although none of the cysteine residues at the thioredoxin-like domain is required for rescue of the grx5 phenotypes. We have concluded that dithiol glutaredoxins are functionally divergent from monothiol ones, but the latter can inter- change their biological activities when compartment barriers are surpassed.