E. V. Mikhailova

Prion-associated proteins in yeast: comparative analysis of isogenic [PSI(+)] and [psi(-)] strains.

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. Copyright

The interaction of dipole modifiers with amphotericin-ergosterol complexes. Effects of phospholipid and sphingolipid membrane composition

The influence of agents, known to affect the membrane dipole potential, phloretin and RH 421, on the multi channel activity of amphotericin B in lipid bilayers of various compositions, was studied. It was shown that the effects were dependent on the membrane’s phospholipid and sphingolipid type. Phloretin enhanced amphotericin B induced steady-state transmembrane current through bilayers made from binary mixtures of POPC (DOPC) and ergosterol and ternary mixture of DPhPC, ergosterol and stearoylphytosphingosine. RH 421 increased steady-state polyene induced transmembrane current through membranes made from binary mixtures of DPhPC (DPhPS) and ergosterol and ternary mixture of DPhPS, ergosterol and stearoylphytosphingosine. It was proposed that the observed effects reflect the fine balance of the interactions between the various components present: amphotericin B, ergosterol, phospholipid, sphingolipid and dipole modifier. The shape of lipid molecules seems to be an important factor impacting the responses of amphotericin B modified bilayers to dipole modifiers. The influence of different phospholipids and sphingolipids on the physical and structural properties of ordered lipid microdomains, enriched in AmB, was also discussed. It was also shown that RH 421 enhanced the antifungal activity of amphotericin B in vitro.

The effect of red pigment on the amyloidization of yeast proteins

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. Copyright

The impact of manipulations with cytoplasmically inherited factors on nuclear transmission and degradation in yeast heterokaryons

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.

Effect of red pigment on amyloidization of yeast

Amyloid-bound thioflavin T fluorescence was studied in lysates of yeast strains that carry mutations in the ADE1 or ADE2 genes and accumulate red pigment as a result of the polymerization of aminoimidazole ribotide (an intermediate of adenine biosynthesis). The fluorescence is drastically enhanced in cells grown in media with high concentrations of adenine (100 mg/l), which suppresses the accumulation of red pigment. Mutations that block the first stages of purine biosynthesis de novo also impede the accumulation of red pigment and produce the same effect on thioflavin fluorescence. Mutations in ADE1 or ADE2 genes in originally white prototrophic strains considerably suppress fluorescence. The fraction of protein polymers was studied by agarose gel electrophoresis, which permitted us to conclude that reduced fluorescence intensity was associated with decreased amyloid content in cells that accumulate red pigment. Model experiments with insulin fibers demonstrate that red pigment binds fibrils and blocks their interaction with thioflavin T. A comparison of lysate pellet proteins from red and white isogenic strains separated by 2D electrophoresis followed by MALDI analysis allowed us to identify 23 pigment-dependent proteins. These proteins mostly belong to functional classes of chaperones and proteins involved in glucose metabolism, which closely correspond to the prion-dependent proteins that we characterized previously. We suppose that the binding of red pigment with amyloid fibrils prevents the generation of prion aggregates and impedes prion propagation by blocking fibril contact with chaperones.

Bud selection and apoptosis-like degradation of nuclei in yeast heterokaryons : a KAR1 effect

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 × KAR1 crosses of two different types (four α KAR1 × akar1 and five α kar1 × aKAR1 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).

Yeast red pigment modifies Amyloid beta growth in Alzheimer disease models in both Saccharomyces cerevisiae and Drosophila melanogaster

Abstract The effect of yeast red pigment on amyloid-β (Aβ) 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 Aβ-GFP aggregates. Yeast cells containing less red pigment had more Aβ-GFP aggregates despite the lower level of overall GFP fluorescence. Western blot analysis with anti-GFP, anti-Aβ and A11 antibodies also revealed that red cells contained a considerably lower amount of Aβ 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 Aβ fibril growth. Transgenic flies expressing Aβ were cultivated on medium containing red and white isogenic yeast strains. Flies cultivated on red strains had a significant decrease in Aβ accumulation levels and brain neurodegeneration. They also demonstrated better memory and learning indexes and higher locomotor ability.

Structural and functional characteristics of various forms of red pigment of yeast Saccharomyces cerevisiae and its synthetic analog

Structural and functional characteristics of the yeast red pigment (product of polymerization of N1-(β-D-ribofuranosyl)-5-aminoimidazole), isolated from ade1 mutant cells of Saccharomyces cerevisiae and its deribosylated derivatives (obtained by acid hydrolysis) and its synthetic pigment analogue (product of polymerization of N1-methyl-5-aminoimidazole in vitro) were obtained. Products of in vitro polymerization were identified using mass spectrometry. The ability of these pigments to inhibit amyloid formation using insulin fibrils was compared. All the studied compounds are able to interact with amyloids and inhibit their growth. Electron and atomic force microscopy revealed a common feature inherent in the insulin fibrils formed in the presence of these compounds—they are merged into conglomerates more stable and resistant to the effects of ultrasound than are insulin aggregates grown without pigments. We suggest that all these compounds can cause coalescence of fibrils partially blocking the loose ends and, thereby, inhibit attachment of monomers and formation of new fibrils.

Effect of red pigment on insulin fibril formation in vitro

The influence of red pigment isolated from yeast Saccharomyces cerevisiae and its low molecular weight derivate on insulin amyloid fibril formation in vitro was studied. The red pigment derivative, which presumably lacked phosphoribosyl moiety due to acid hydrolysis, retained the ability to inhibit fluorescence intensity (FI) of amyloid bound Thioflavine T. It was found that FI inhibition depended on the concentration of both pigment forms. Both forms were also able to compete with Thioflavine T for amyloid fibril binding. Electron microscopy revealed that fibrils reduced in size in the presence of red pigment.

Comparative assay of amyloid and prion contents in yeast cells

Amyloid contents were quantitatively assayed in crude yeast lysates treated with thioflavin T that specifically stained amyloid fibrils. We demonstrated that guanidine hydrochloride (GuHCl) treatment and overexpression of Hsp104p chaperone resulted in the elimination of the [PSI+] factor and that the stable decline in amyloid contents followed from the reduced fluorescence intensity (IF) of thioflavin T. Overexpression of the SUP35 gene coding the protein prionizable to [PSI+] results in the generation of [PSI+] clones with increased thioflavin T IF. Transmission of [PSI+] factor by cytoduction in crossings of recipients with low IF was also accompanied by stable IF enhancement in cytoductants, indicating enriched amyloid contents. Thus, in model experiments, modifying the quantity of [PSI+] factor, a yeast prion amyloid, the change in thioflavin T IF corresponds to the expected shift in amyloid contents, the IF shift behaving as a cytoplasm hereditary determinant. It is concluded that thioflavin T IF allows for the quantitative estimation of amyloid contents in cells. The stable mitotic IF shift induced by agents affecting the prion composition permits the quantitative evaluation of prion contribution into amyloid pool. It is possible to assume that the monitoring of thiophlavin T IF shifts under the exposure of agents affecting prion pattern may be helpful to disclose previously unknown prions in yeast strains.