RÉSUMÉ
Estimating the amyloid level in yeast Saccharomyces, we found out that the red pigment (product of polymerization of aminoimidazole ribotide) accumulating in ade1 and ade2 mutants leads to drop of the amyloid content. We demonstrated in vitro that fibrils of several proteins grown in the presence of the red pigment stop formation at the protofibril stage and form stable aggregates due to coalescence. Also, the red pigment inhibits reactive oxygen species accumulation in cells. This observation suggests that red pigment is involved in oxidative stress response. We developed an approach to identify the proteins whose aggregation state depends on prion (amyloid) or red pigment presence. These sets of proteins overlap and in both cases involve many different chaperones. Red pigment binds amyloids and is supposed to prevent chaperone-mediated prion propagation. An original yeast-Drosophila model was offered to estimate the red pigment effect on human proteins involved in neurodegeneration. As yeast cells are a natural feed of Drosophila, we could compare the data on transgenic flies fed on red and white yeast cells. Red pigment inhibits aggregation of human Amyloid beta and α-synuclein expressed in yeast cells. In the brain of transgenic flies, the red pigment diminishes amyloid beta level and the area of neurodegeneration. An improvement in memory and viability accompanied these changes. In transgenic flies expressing human α-synuclein, the pigment leads to a decreased death rate of dopaminergic neurons and improves mobility. The obtained results demonstrate yeast red pigment potential for the treatment of neurodegenerative diseases.
Sujet(s)
Amyloïdose , Prions , Amyloïde/métabolisme , Peptides bêta-amyloïdes/métabolisme , Animaux , Animal génétiquement modifié , Drosophila , Prions/métabolisme , Agrégats de protéines , Saccharomyces cerevisiae/métabolisme , alpha-Synucléine/métabolismeRÉSUMÉ
The effect of yeast red pigment on amyloid-ß (Aß) aggregation and fibril growth was studied in yeasts, fruit flies and in vitro. Yeast strains accumulating red pigment (red strains) contained less amyloid and had better survival rates compared to isogenic strains without red pigment accumulation (white strains). Confocal and fluorescent microscopy was used to visualise fluorescent Aß-GFP aggregates. Yeast cells containing less red pigment had more Aß-GFP aggregates despite the lower level of overall GFP fluorescence. Western blot analysis with anti-GFP, anti-Aß and A11 antibodies also revealed that red cells contained a considerably lower amount of Aß GFP aggregates as compared to white cells. Similar results were obtained with exogenous red pigment that was able to penetrate yeast cells. In vitro experiments with thioflavine and TEM showed that red pigment effectively decreased Aß fibril growth. Transgenic flies expressing Aß were cultivated on medium containing red and white isogenic yeast strains. Flies cultivated on red strains had a significant decrease in Aß accumulation levels and brain neurodegeneration. They also demonstrated better memory and learning indexes and higher locomotor ability.
Sujet(s)
Maladie d'Alzheimer/métabolisme , Peptides bêta-amyloïdes/métabolisme , Saccharomyces cerevisiae/métabolisme , Maladie d'Alzheimer/anatomopathologie , Animaux , Technique de Western , Encéphale/métabolisme , Encéphale/anatomopathologie , Drosophila melanogaster , Cytométrie en flux , Activité motrice/physiologie , Fragments peptidiques/métabolisme , Réaction de polymérisation en chaine en temps réel , Saccharomyces cerevisiae/pathogénicitéRÉSUMÉ
The intensity of amyloid-bound thioflavine T fluorescence was studied in crude lysates of yeast strains carrying mutations in the ADE1 or ADE2 genes and accumulating the red pigment (a result of polymerization of aminoimidazoleribotide), and in white isogenic strains--either adenine prototrophs or carrying mutations at the first stages of purine biosynthesis. We found that the red pigment leads to a drop of amyloid content. This result, along with the data on separation of protein polymers of white and red strains in PAGE, suggests that the red pigment inhibits amyloid fibril formation. The differences in transmission of the thioflavine T fluorescence pattern by cytoduction and in blot-hybridization of pellet proteins of red and white [PSI(+) ] strains with Sup35p antibodies confirmed this conclusion. Purified red pigment treatment also led to a decrease of fluorescence intensity of thioflavine T bound to insulin fibrils and to yeast pellet protein aggregates from [PSI(+) ] strains. This suggests red pigment interaction with amyloid fibrils. Comparison of pellet proteins from red and white isogenic strains separated by 2D-electrophoresis followed by MALDI analysis has allowed us to identify 48 pigment-dependent proteins. These proteins mostly belong to functional classes of chaperones and proteins involved in glucose metabolism, closely corresponding to prion-dependent proteins that we characterized previously. Also present were some proteins involved in stress response and proteolysis. We suppose that the red pigment acts by blocking certain sites on amyloid fibrils that, in some cases, can lead in vivo to interfere with their contacts with chaperones and the generation of prion seeds.
Sujet(s)
Amyloïde/composition chimique , Amyloïde/métabolisme , Pigments biologiques/métabolisme , Protéines de Saccharomyces cerevisiae/composition chimique , Protéines de Saccharomyces cerevisiae/métabolisme , Saccharomyces cerevisiae/métabolisme , Thiazoles/métabolisme , Amyloïde/génétique , Benzothiazoles , Régulation négative , Données de séquences moléculaires , Liaison aux protéines , Saccharomyces cerevisiae/composition chimique , Saccharomyces cerevisiae/génétique , Protéines de Saccharomyces cerevisiae/génétique , Spectrométrie de masse MALDIRÉSUMÉ
A large group of prion-associated proteins was identified in yeast cells using a new approach, comparative analysis of pellet proteins of crude cell lysates in isogenic strains of Saccharomyces cerevisiae differing by their prion composition. Two-dimensional (2D) electrophoresis followed by MALDI analysis of the pellet proteins of [PSI(+)] and [psi(-)] strains after prion elimination by GuHCl and prion transmission by cytoduction permitted identification of ca. 40 proteins whose aggregation state correlated with the change of prion(s) content. Approximately half of these proteins belonged to chaperones and to enzymes of glucose metabolism. Chaperones are known to be involved in prion metabolism and are expected to be present in prion-containing aggregates, but glucose metabolism enzymes are not predicted to be present. Nevertheless, several recent data suggest that their presence is not incidental. We detected six proteins involved in oxidative stress response and eight in translation. Also notable is a protease. Most of the identified proteins seem to be prion-associated, but we cannot exclude the possibility that several proteins may propagate as prions.
Sujet(s)
Protéines fongiques/isolement et purification , Protéines fongiques/métabolisme , Prions/isolement et purification , Prions/métabolisme , Saccharomyces cerevisiae/composition chimique , Saccharomyces cerevisiae/métabolisme , Électrophorèse bidimensionnelle sur gel , Liaison aux protéines , Spectrométrie de masse MALDIRÉSUMÉ
It has been shown that defects in cell fusion during mating can trigger programmed cell death in the yeast Saccharomyces cerevisiae. We wished to test whether defects in nuclear migration during cell fusion have the same effect. A partial pedigree analysis of nine kar1 x KAR1 crosses of two different types (four alpha KAR1 x a kar1 and five alpha kar1 x a KAR1 crosses) was carried out, and quantitative estimates of the frequencies of different mother/daughter (m/d) classes were obtained. The kar1 mutation affects nuclear congression and delays nuclear fusion. In each cross tested, the nucleus that entered the first bud tended to be the one contributed by the cell that carried the wild-type allele of KAR1. If budding was delayed by nutrient limitation, the kar1 nucleus could be rescued, indicating that the primary effect of the kar1 mutation is that it slows spindle action. Many m/d classes appear as a result of the degradation of one of the nuclei in the heterokaryon. Loss of nuclei in heterokaryons was accompanied by an accumulation of reactive oxygen species (ROS), and by abnormalities in nuclear structure revealed by TUNEL (terminal transferase-mediated dUTP nick end-labeling) analysis, DAPI staining and by histone-GFP fluorescence patterns which suggested an apoptosis-like process. Often only one nucleus was degraded, and ROS accumulation was restricted to one half of the zygote. We therefore suggest that the data obtained can be explained by apoptosis-like death of a half-cell (cell body).
Sujet(s)
Protéines nucléaires/génétique , Protéines nucléaires/physiologie , Protéines de Saccharomyces cerevisiae/génétique , Protéines de Saccharomyces cerevisiae/physiologie , Saccharomyces cerevisiae/génétique , Saccharomyces cerevisiae/physiologie , Allèles , Apoptose , Fusion cellulaire , Noyau de la cellule/métabolisme , Survie cellulaire , Croisements génétiques , ADN/composition chimique , Protéines fongiques/composition chimique , Gènes fongiques , Génotype , Protéines à fluorescence verte/composition chimique , Histone/composition chimique , Méthode TUNEL , Indoles/pharmacologie , Modèles génétiques , Mutation , Pedigree , Espèces réactives de l'oxygène , Facteurs tempsRÉSUMÉ
Heterokaryotic zygotes in yeast provide a unique possibility to study the survival and transmission of two genetically diverse nuclei in one cell. Using partial pedigree analysis, we show that various treatments used to change cytoplasmic hereditary determinants can essentially affect nuclear transmission in yeast heterokaryons. This includes choice of nucleus to enter the first bud and incidence of various classes of mother/daughter pairs demonstrating nuclear degradation patterns in heterokaryotic zygotes. These treatments include guanidine hydrochloride, a prion-curing agent, ethidium bromide, an agent causing elimination of mitochondrial DNA, and cytoplasm replacement by cytoduction, which leads to mtDNA replacement and transfer of some other cytoplasmically inherited determinants. The genetic and cytological evidence obtained favors prion involvement in nuclear transmission and suggests apoptotic features in nuclear degradation in yeast heterokaryotic zygotes.
Sujet(s)
Noyau de la cellule/composition chimique , Noyau de la cellule/métabolisme , Cytoplasme/métabolisme , Techniques génétiques , Saccharomyces cerevisiae/physiologie , Apoptose , ADN mitochondrial/composition chimique , Éthidium/pharmacologie , Protéines fongiques/composition chimique , Guanidine/composition chimique , Guanidine/pharmacologie , Haploïdie , Intercalants/pharmacologie , Microscopie de fluorescence , Phénotype , Prions/composition chimique , Espèces réactives de l'oxygène , Saccharomyces cerevisiae/métabolisme , Zygote/composition chimique , Zygote/métabolismeRÉSUMÉ
The integrative vector pPIC3 for the yeast Pichia pastoris and a cDNA fragment encoding a fusion protein consisting of green fluorescent protein (GFP) and actin 5C of the fruit fly Drosophila melanogaster were used to construct a pPIC3-GFP-actin 5C expression plasmid. The P. pastoris host strain GS115 was transformed with the pPIC3-GFP-actin 5C carrying HIS4 as a selective marker. The transformants were selected on a histidine-deficient medium, and were shown to contain the gene of GFP-actin 5C fusion protein. Expression was induced by cultivation of the transformant cells in a methanol-containing medium. Production of the fusion protein in the yeast was detected by the bright green fluorescence of the GFP tag. The pattern of yeast cytoskeleton labeling by the fusion indicated proper folding and functioning of GFP-actin 5C in a heterologous system in vivo. After cell destruction, purification of GFP-actin 5C was performed by DNase I-Sepharose. Efficient binding of the chimera to the DNase I indicated nativity of the actin 5C fusion in vitro. SDS electrophoresis and further Western blot confirmed the purified protein to exhibit the expected molecular mass of about 70 kDa. The recombinant GFP-actin 5C was used to produce polyclonal antibodies, which had not been reported so far but are extremely needed for immuno-labeling and isolation of wild-type and mutant forms of actin 5C.
Sujet(s)
Actines/biosynthèse , Pichia/métabolisme , Actines/composition chimique , Actines/génétique , Actines/isolement et purification , Technique de Western , Clonage moléculaire , Expression des gènes , Protéines à fluorescence verte , Protéines luminescentes/génétique , Protéines luminescentes/métabolisme , Pichia/génétique , Plasmides/métabolisme , Protéines de fusion recombinantes/biosynthèse , Protéines de fusion recombinantes/métabolismeRÉSUMÉ
We have found that cells derived from heterokaryons (HK) showing phenotypical traits, coded by the nucleus of one parental strain and by the cytoplasm of the other, may produce mitotic progeny in which the second nucleus is apparently present but not expressed. This 'concealed' nucleus can be forced to expression after growth of these cytoductants on proper selective media. Using a micromanipulator, the buds containing both parental nuclei were isolated in various crosses. Cloning these HK from a rich medium (YEPD) revealed that nearly all of them were composed of a mixture of hybrid cells and cells of one of the parents. Cells of the other parent were present in a very small proportion, if detectable at all. We showed that the percentage of concealed HK decreases when limiting the growth of the strain that serves as a donor of the concealed nucleus. Consequently, our explanation for the presence of concealed nuclei in HK is the low production rate of daughter cells containing both nuclei, which accounts for the lack of a visible phenotype in HK, together with the low replication rate (or fast nuclease degradation) of one of the nuclei. In homosexual crosses, selection allows us to switch the concealed nucleus to normal replication rate. Some abnormalities of meiosis due to hidden nuclei are shown.