Neurodegenerative amyloidoses: the yeast model

More than 20 human diseases are related to protein misfolding which causes formation of amyloids, fibrillar aggregates of normally soluble proteins. Such diseases are called amyloid diseases or amyloidoses. Of them only prion diseases are transmissible. Amyloids of the prion type are described in lower eukaryotes. However, in contrast to mammalian prions, which cause incurable neurodegenerative diseases, prions of lower eukaryotes are related to some non-chromosomally inherited phenotypic traits. Here we summarize the results of studies of prions of the yeast Saccharomyces cerevisiae and of the use of yeast model for investigation of some human amyloidoses, such as prion diseases, Alzheimer's, Parkinson's, and Huntington's diseases.     

Rnq1 protein protects [<Emphasis Type="Italic">PSI</Emphasis><Superscript>+</Superscript>] prion from effect of the <Emphasis Type="Italic">PNM</Emphasis> mutation

The interaction of [PSI ⁺] and [PIN ⁺] factors in yeast Saccharomyces cerevisiae is known as the first evidence of prions networks. In [PIN ⁺] cells, Rnq1p prion aggregates work as a template for Sup35p aggregation, which is essential for [PSI ⁺] induction. No additional factors are required for subsequent Sup35p aggregation. Nevertheless, several recent reports provide data that indicate a more complex interplay between these prions. Our results show that the presence of Rnq1p in the cell significantly decreases the loss of [PSI ⁺] prion, which is caused by a double mutation in SUP35 (Q61K, Q62K substitutions in the Sup35 protein). These observations support the existence of interaction networks that converge on a strong linkage of prionogenic and prion-like proteins, and the participation of Rnq1 protein in the maintenance of prion [PSI ⁺].     

A glutamine/asparagine-rich fragment of Gln3, but not the full-length protein,aggregates in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

The amino acid sequence of protein Gln3 in yeast Saccharomyces cerevisiae has a region enriched with Gln (Q) and Asn (N) residues. In this study, we analyzed the effects of overexpression of Gln3 and its Q/N-rich fragment fused with yellow fluorescent protein (YFP). Being overexpressed, full-length Gln3-YFP does not form aggregates, inhibits vegetative growth, and demonstrates nuclear localization, while the Q/N-rich fragment (Gln3QN) fused with YFP forms aggregates that do not colocalize with the nucleus and do not affect growth of the cells. Although detergent-resistant aggregates of Gln3QN are formed in the absence of yeast prions, the aggregation of Gln3QN significantly increases in the presence of [PIN⁺] prion, while in the presence of two prions, [PSI⁺] and [PIN⁺], the percentage of cells with Gln3QN aggregates is significantly lower than in the strain bearing only [PIN⁺]. Data on colocalization demonstrate that this effect is mediated by interaction between Gln3QN aggregates and [PSI⁺] and [PIN⁺] prions.     

<Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> strains associated with the production of cachaça: identification and characterization by traditional and molecular methods (PCR,PFGE and mtDNA-RFLP)

A study of the genetic diversity of populations of Saccharomyces cerevisiae was conducted in ten different cachaça producers (alambiques) in the southern state of Minas Gerais, Brazil. A total of 106 isolates were identified by PCR using the primer SCREC114, specific to S. cerevisiae, by pulsed-field gel electrophoresis (PFGE) and by restriction fragment polymorphism of mitochondrial DNA analysis (RFLP-mtDNA). PCR showed a product of amplification to 61 isolates, enabling a rapid identification of S. cerevisiae in different alambiques. Nine different profiles were found by PFGE; all the yeasts identified as S. cerevisiae by PCR had profiles similar to that of the marker S. cerevisiae, highlighting the specificity of primer SCREC114. RFLP-mtDNA, using four different enzymes, enabled the grouping of strains of S. cerevisiae, with 80%–100% similarity. Some alambiques that had a higher frequency of S. cerevisiae characterized by PCR and PFGE, had a lower level of genetic diversity determined by RFLP-mtDNA, indicating the ability of these strains to lead the fermentative process.     

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.     

The First Report on Single Nucleotide Polymorphisms of the <Emphasis Type="Italic">HSPA13</Emphasis> Gene in Koreans

Heat shock proteins (HSPs) are known as molecular chaperones, and they function in response to cell stress. HSPA13, also called STCH, is a member of the HSP70 family. In general, HSP70 family may play a protective role in prion diseases. In a recent study, the overexpression of HSPA13 was shown to shorten the incubation time of prion diseases. Although the exact role of HSPA13 in the pathogenesis of prion diseases remains unknown, the expression level of HSPA13 is significantly associated with the latent phase of prion diseases. It has been known that single nucleotide polymorphisms (SNPs) in promoter and open reading frame (ORF) region of genes can affect either gene expression or gene function. The purpose of this study was to investigate genotype and allele frequencies of SNPs found in the promoter and ORF of HSPA13 in healthy Korean population to obtain the information for subsequent population genetics and prion diseases studies. We observed four SNPs in the promoter region of HSPA13, of which two have previous identified (c.-608C>G; rs2242662 and c.-381G>A; rs2242661) and two are novel (c.-321C>T and c.-300A>G). Interestingly, we did not observe any polymorphisms in the ORF of this gene. To our knowledge, this is the first study of polymorphisms in the human HSPA13 gene.     

The genusSaccharomyces (Meyen) Reess

The authors submit a taxonomic evaluation of an intermediate group of strains between the speciesSaccharomyces carlsbergensis Hansen andSaccharomyces cerevisiae Hansen. The material consisted of atypical strains of “bottom” brewer’s yeasts and the synonymous strainsSaccharomyces monacensis Hansen andSaccharomyces mandshuricus Saito. It was found that there were two different serological types in the speciesSaccharomyces carlsbergensis, one of which was characterized by the presence of antigen “C” and was typical for this species, while the other possessed antigen “M” and was grouped roundSaccharomyces monacensis. This second serological type merges with a group of strains which gives only one third fermentation of raffinose, so that it is actually an intermediate betweenSaccharomyces cerevisiae Hansen andSaccharomyces carlsbergensis Hansen and indicates the course of progressive development from the former species to the latter. No close similarity was found betweenSaccharomyces mandshuricus Saito and some of the strains of the transitional group or typical representatives of the two main species, and the authors therefore consider that there is some obscurity as to its synonymity withSaccharomyces carlsbergensis.     

Search for genes influencing the maintenance of the [<Emphasis Type="Italic">ISP</Emphasis><Superscript>+</Superscript>] prion-like antisuppressor determinant in yeast with the use of an insertion gene library

The prion-like determinant [ISP ⁺] manifests itself as an antisuppressor of certain sup35 mutations. To establish that [ISP ⁺] is indeed a new yeast prion, it is necessary to identify the gene that codes for the protein whose prion form is [ISP ⁺]. Analysis of the transformants obtained by transformation of an [ISP ⁺] strain with an insertion gene library revealed three genes controlling the [ISP ⁺] maintenance: UPF1, UPF2, and SFP1. SFP1 codes for a potentially prionogenic protein, which is enriched in Asn and Gln residues, and is thereby the most likely candidate for the [ISP ⁺] structural gene. UPF1 and UPF2 code for components of nonsense-mediated mRNA decay. The [ISP ⁺] elimination caused by UPF1 and UPF2 inactivation was reversible, and Upf1p and Upf2p were not functionally related to phosphatase Ppz1p, which influences the [ISP ⁺] manifestation. Possible mechanisms sustaining the influence of UPF1 and UPF2 on [ISP ⁺] maintenance are discussed.     

Distinct mechanisms of phenotypic effects of inactivation and prionization of Swi1 protein in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Prions are proteins that under the same conditions can exist in two or more conformations, and at least one of the conformations has infectious properties. The prionization of a protein is typically accompanied by its functional inactivation due to sequestration of monomers by the prion aggregates. The most of prions has been identified in the yeast Saccharomyces cerevisiae. One of them is [SWI ⁺], a prion isoform of the Swi1 protein, which is a component of the evolutionarily conserved chromatin remodeling complex SWI/SNF. Earlier, it was shown that the prionization of [SWI ⁺] induces a nonsense suppression, which leads to weak growth of the [SWI ⁺] strains containing mutant variants of the SUP35 gene and the nonsense allele ade1-14 _UGA on selective medium without adenine. This effect occurs because of [SWI ⁺] induction that causes a decrease in the amount of the SUP45 mRNA. Strains carrying the SWI1 deletion exhibit significantly higher suppression of the ade1-14 _UGA nonsense mutation than the [SWI ⁺] strains. In the present study, we identified genes whose expression is altered in the background of the SWI1 deletion using RNA sequencing. We found that the ade1-14 _UGA suppression in the swi1Δ strains is caused by an increase in the expression of this mutant allele of the ADE1 gene. At the same time, the SUP45 expression level in the swi1Δ strains does not significantly differ from the expression level of this gene in the [swi ^–] strains. Thus, we have shown that the phenotypic effects of Swi1 prionization and deletion are mediated by different molecular mechanisms. Based on these data, we have concluded that the prionization of proteins is not only unequal to their inactivation, but also can lead to the acquisition of novel phenotypic effects and functions.     

Identification of yeasts during alcoholic fermentation of <Emphasis Type="Italic">tchapalo</Emphasis>, a traditional sorghum beer from Côte d’Ivoire

This study investigated the diversity and dynamics of yeasts involved in alcoholic fermentation of a traditional sorghum beer from Côte d’Ivoire, tchapalo. A total of 240 yeast strains were isolated from fermenting sorghum wort inoculated with dry yeast from two geographic regions of Côte d’Ivoire (Abidjan and Bondoukou). Initial molecular identification to the species level was carried out using RFLP of PCR-amplified internal transcribed spacers of rDNA (ITS1-5.8S-ITS2). Ten different profiles were obtained from the restriction of PCR products with the three endonucleases HaeIII, CfoI and HinfI. Sequence analysis of the D1/D2 domain of the 26S rDNA and the ACT1 gene allowed us to assign these groups to six different species: Saccharomyces cerevisiae-like, Candida tropicalis, Pichia kudriavzevii, Pichia kluyveri, Kodamaea ohmeri and Meyerozyma caribbica. The most frequent species associated with tchapalo fermentation was S. cerevisiae-like (87.36%), followed by C. tropicalis (5.45%) and M. caribbica (2.71%). S. cerevisiae-like strains were diploid heterozygotes and exhibited three to four nucleotides divergence from the type strain in the D1/D2 domain and several indels in the more discriminant sequence of the intron of the ACT1 gene. During the process, the yeast species isolated and their frequencies varied according to the geographic origin of the dry yeast. The occurrence of some species was sporadic and only two non-Saccharomyces species were found in the final product.     

Bolete Productivity of Cistaceous Scrublands in Northwestern Spain<Superscript>1</Superscript>

Bolete Productivity of Cistaceous Scrublands in Northwestern Spain. Cistaceous scrublands dominated by Cistus ladanifer and Halimium lasianthum are widely distributed in northwestern Spain and are known to harbor the popular edible mushrooms Boletus edulis and B. aereus. As commercial harvest of these species is an important activity in Spain’s forests, in some cases generating greater revenue than timber production, we tried to determine whether there is commercial potential for bolete harvest in young cistaceous scrublands. Transects of 50 m × 2 m were randomly established in cistaceous scrublands and bolete yield was measured. High production of these valuable mushrooms was associated with 3-year-old plants, a much earlier onset than observed for Pinus and Quercus stands. Management recommendations for the studied scrub ecosystems are proposed that take into account the economic value of the mushrooms.     

New ideas on the system of the Permian <Emphasis Type="Italic">Inoceramus</Emphasis>-like bivalves of the eastern Boreal zone

New concepts of systematics and phylogeny of the Permian Inoceramus-like bivalve mollusks of the eastern part of the Boreal zone are discussed based on analysis of the group’s historical development. All taxa studied are referred to the family Kolymiidae Kusnezov, which is divided into two subfamilies, Kolymiinae and Atomodesmatinae. In the subfamily Kolymiinae, two new genera are described: Praekolymia with the type species P. archboldi sp. nov. and P. urbajtisae sp. nov., and Taimyrokolymia with the type species T. ustritskyi sp. nov. In the subfamily Atomodesmatinae, two new genera, Costatoaphanaia and Okhotodesma, are described. The development of the group is shown to be generally autochthonous with some invasions (genus Atomodesma and, probably, Trabeculatia) from extraboreal regions.     

Clinical relevance of infections with zoonotic and human oral species of <Emphasis Type="Italic">Campylobacter</Emphasis>

Genus Campylobacter has been recognized as a causative bacterial agent of animal and human diseases. Human Campylobacter infections have caused more concern. Campylobacters can be classified into two groups in terms of their original host: zoonotic and human oral species. The major zoonotic species are Campylobacter jejuni and Campylobacter coli, which mostly reside in the intestines of avian species and are transmitted to humans via consumption of contaminated poultry products, thus causing human gastroenteritis and other diseases as sequelae. The other campylobacters, human oral species, include C. concisus, C. showae, C. gracilis, C. ureolyticus, C. curvus, and C. rectus. These species are isolated from the oral cavity, natural colonization site, but have potential clinical relevance in the periodontal region to varying extent. Two species, C. jejuni and C. coli, are believed to be mainly associated with intestinal diseases, but recent studies suggested that oral Campylobacter species also play a significant role in intestinal diseases. This review offers an outline of the two Campylobacter groups (zoonotic and human oral), their virulence traits, and the associated illnesses including gastroenteritis.     

Internal initiation of translation of mRNA in the methylotrophic yeast <Emphasis Type="Italic">Hansenula polymorpha</Emphasis>

Besides regular cap-dependent translation of mRNA, eukaryotes exploit internal initiation of translation driven by internal ribosome entry sites (IRESs). It is supposed that internal initiation provides translation of cellular mRNAs under stress conditions where the cap-dependent initiation is reduced. A number of IRESs have been characterized in mammalian mRNAs, but only a few examples are known in lower eukaryotes, particularly in yeasts. Here we identified two IRESs in the thermotolerant methylotrophic yeast Hansenula polymorpha DL-1. These sites are located in 5′-untranslated regions of genes HPODL_02249 and HPODL_04025 encoding a hypothetical membrane protein and actin-binding protein, respectively. In Saccharomyces cerevisiae cells, both IRESs drive expression of a second gene of a bicistronic mRNA, as well as translation of hairpin-containing monocistronic mRNA. The possibility of spurious splicing or presence of a cryptic promoter in the IRES sequences was ruled out, indicating that expression of a second gene of a bicistronic mRNA was IRESdependent. We evaluated IRES activity of both elements and found that under normal physiological conditions its contribution to the overall translation of the respective mRNAs in yeast cells is about 0.3-0.4%. Therefore, these results suggest that the IRES-dependent translation initiation mechanism exists in Hansenula polymorpha.     

Molecular genetics of type 1 diabetes mellitus: Achievements and future trends

Type 1 diabetes mellitus (T1DM) is a widespread severe disease that results from autoimmune destruction of β cells in Langerhans islets of the pancreas. To date, several loci involved in T1DM have been reliably identified using various approaches: the MHC locus, VNTR within the 5′-nontranscibed region of the insulin gene (INS), CTLA4 (T-cell surface receptor), PTPN22, PTPN2 (T-cell tyrosine phosphatases), IL2 (interleukin 2, IL-2), IL2RA (IL-2 receptor α chain), KIAA0350 (unknown function), and IFIH1 (receptor for double-stranded DNA generated in virus infections). Functional analysis of their protein products confirmed the hypothesis that T1DM is underlain by deregulation of the mechanisms of immune tolerance and, on the other hand, a destructive immune response against the body’s own proteins after virus infection or some other immune stress. Thus the protein products of MHC, INS, PTPN22, and PTPN2 are involved in the intrathymic formation of the T-cell repertoire, responsible for immune defense of the body. On the other hand, nonspecific activation of T cells, which starts autoimmune destruction of pancreatic β cells, is most likely associated with the protein products of CTLA4, IL2, IL2RA, and, possibly, PTPN22 and PTPN2. Apart from the genes with unknown functions, the only exception is IFIH1, but its association with T1DM confirms that certain virus infections can activate autoreactive T cells and lead to T1DM.     

New Approach to Identification of Genes Controlling Cell Wall Biogenesis in the Yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

MCD4 codes for a protein presumably adding the phosphoethanolamine moiety to the first mannose residue of glycosylphosphatidylinositol (GPI) precursors in the yeast Saccharomyces cerevisiae. The role of this modification is still unclear. The phenotypic effects of some MCD4 mutations are probably unrelated to defects in GPI synthesis, suggesting additional functions for Mcd4p. To study the Mcd4p functions in more detail, a search for the genes whose mutations are lethal or semilethal in combination with the ssu21 mutation of MCD4 was performed. Six such mutations were isolated, including some mutations causing sensitivity to SDS and/or calcofluor white. Genes complementing two out of the six mutations were cloned and identified as MNN9, which is involved in the formation of outer chains of N-linked glycans of secreted proteins, and GWT1, which codes for an endoplasmic reticulum protein involved in GPI biosynthesis. In both cases, growth inhibition was probably caused by defective biogenesis of the cell wall and a misfolding of secreted proteins. The proposed approach is suitable for seeking new genes controlling cell wall biogenesis.     

Pistil-function breakdown in a new <Emphasis Type="Italic">S</Emphasis>-allele of European pear, <Emphasis Type="Italic">S</Emphasis><Subscript><Emphasis Type="Italic">21</Emphasis></Subscript>°, confers self-compatibility

European pear exhibits RNase-based gametophytic self-incompatibility controlled by the polymorphic S-locus. S-allele diversity of cultivars has been extensively investigated; however, no mutant alleles conferring self-compatibility have been reported. In this study, two European pear cultivars, ‘Abugo’ and ‘Ceremeño’, were classified as self-compatible after fruit/seed setting and pollen tube growth examination. S-genotyping through S-PCR and sequencing identified a new S-RNase allele in the two cultivars, with identical deduced amino acid sequence as S ₂₁ , but differing at the nucleotide level. Test-pollinations and analysis of descendants suggested that the new allele is a self-compatible pistil-mutated variant of S ₂₁ , so it was named S ₂₁ °. S-genotypes assigned to ‘Abugo’ and ‘Ceremeño’ were S ₁₀ S ₂₁ ° and S ₂₁ °S ₂₅ respectively, of which S ₂₅ is a new functional S-allele of European pear. Reciprocal crosses between cultivars bearing S ₂₁ and S ₂₁ ° indicated that both alleles exhibit the same pollen function; however, cultivars bearing S ₂₁ ° had impaired pistil-S function as they failed to reject either S ₂₁ or S ₂₁ ° pollen. RT-PCR analysis showed absence of S ₂₁ °-RNase gene expression in styles of ‘Abugo’ and ‘Ceremeño’, suggesting a possible origin for S ₂₁ ° pistil dysfunction. Two polymorphisms found within the S-RNase genomic region (a retrotransposon insertion within the intron of S ₂₁ ° and indels at the 3′UTR) might explain the different pattern of expression between S ₂₁ and S ₂₁ °. Evaluation of cultivars with unknown S-genotype identified another cultivar ‘Azucar Verde’ bearing S ₂₁ °, and pollen tube growth examination confirmed self-compatibility for this cultivar as well. This is the first report of a mutated S-allele conferring self-compatibility in European pear.     

Biological functions of amyloids: Facts and hypotheses

Amyloids are fibrous protein aggregates that arise via polymerization of proteins with their concurrent conformational rearrangement and the formation of a specific cross-β structure. Amyloids are of particular interest as a cause of a vast group of human and animal diseases called amyloidoses. Some of these diseases are caused by prions, a specific type of amyloids, and are transmissible. Apart from mammals, prion amyloids are described in lower eukaryotes, where they act as nonchromosomal genetic determinants. Although amyloids are usually associated with pathologies in humans and animals, the increasing number of findings suggests that the acquisition of an amyloid or prion form by a protein is of biological significance in some cases. The review summarizes the data on the biological significance of prion and nonprion amyloids in a wide range of species from bacteria to mammals.