A. V. Artemov

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

Detection of Concealed “Illegitimate” Nuclei in Tetrad Analysis of the Diploid Progeny of Heterokaryons in Saccharomyces cerevisiae

In this work, the studies on the previously detected phenomenon of concealed heterokaryosis in Saccharomyces cerevisiaewere continued. In genetic and Southern blotting experiments, one of the nuclei in the heterokaryon was shown to be active (capable of division and ensuring the corresponding cell phenotype), whereas the other was not expressed until the heterokaryotic clone was transferred to the medium selective for this concealed nucleus. Moreover, the concealed nucleus was able to assume the active state after fusion with the second parental nucleus. It was analyzed whether the nuclei with new marker combinations occurring in meiosis can behave as exceptional nuclei. Tetrad analysis of hybrids carrying the kar1mutation in their nuclei revealed the relatively high percentage of exceptional tetrads (more than 10%). One spore in these tetrads usually formed diploid cells capable of sporulation. The presented data of genetic and molecular biological studies testify in favor of the assumption that abnormal spores contain two nuclei, which form an “illegitimate” hybrid after fusion. An extraneous nucleus (termed x) has usually a genotype close to that of one of the spores in this tetrad. Thus, it was assumed that the additional DNA replication round occurs in the absence of cell division during one of meiotic divisions. Results of cytological analysis conducted by the method of specific DNA staining confirmed the existence of exceptional tetrads, one spore of which contains two nuclei.

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

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.

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.

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.

Comparison of crude lysate pellets from isogenic strains of yeast with different prion composition: Identification of prion-associated proteins

A new approach involving the comparative analysis of proteins of crude cell lysate pellets from isogenic strains of Saccharomyces cerevisiae distinguished by their prion composition permitted us to identify a large group of prion-associated proteins in yeast cells. 35 proteins whose aggregation state depends on prion content have been identified by 2D-electrophoresis followed by the MALDI analysis of a recipient [psi−] strain and of [PSI+] cytoductant. Approximately half of these proteins belong to functional groups of chaperones and enzymes involved in glucose metabolism. Other proteins are involved in translation, stress response and protein degradation. The data obtained are compared with the results of other groups who used different approaches to detect proteins involved in prion aggregates.

The effect of yeast Saccharomyces cerevisiae red pigment on the expression of cloned human α-synuclein

Two transgenic yeast strains expressing human α-synuclein were used to study the impact of yeast red pigment exhibiting antiamyloid properties. It has been demonstrated that the endogenous red pigment produced under special conditions in strains carrying an ade1 mutation inhibits the expression of the hybrid protein α-synuclein-GFP. This was evident from the reduced mean value of GFP fluorescence and diminished number of cells accumulating cytoplasmic inclusions of α-synuclein-GFP. Exogenous forms of the purified red pigment (natural, synthetic and hydrolyzed derivatives) differ from the endogenous red pigment by their effect on α-synuclein. Exogenous red pigments increased the number of both cells expressing GFP fluorescence and those containing cytoplasmic inclusions. However, both endogenous and exogenous red pigments reduced the cloned α-synuclein toxicity and resulted in redistribution of the α-synuclein in cells. α-Synuclein content decreased in cell lysate pellets and increased in supernatants.

Expression of Recombinant Actin 5C from Drosophila in the Methylotrophyc Yeast Pichia Pastoris

A system of expression for the foreign actin gene in yeast cells Pichia pastoris has been developed. As a target protein, the Drosophila cytoplasmic actin 5C, which has 90% homology to the β-actin of higher eukaryotes, was used. In the present work, in order to develop conditions for biosynthesis of the target protein in yeast cells and a purification procedure for the recombinant protein, a GFP-actin fusion protein containing green fluorescent protein (GFP) as a fusion tag was expressed and purified. The size and survival of P. pastoris cells producing recombinant protein were characterized and shown to depend on the accumulation of recombinant actin. The purified fusion protein was used to obtain a polyclonal antibody necessary for testing for recombinant actin.