Saccharomyces cerevisiae prion [PSI ] is a self‐propagating isoform of the eukaryotic release factor eRF3 (Sup35p). Sup35p consists of the evolutionary conserved release factor domain (Sup35C) and two evolutionary variable regions – Sup35N, which serves as a prion‐forming domain in S. cerevisiae, and Sup35M. Here, we demonstrate that the prion form of Sup35p is not observed among industrial and natural strains of yeast. Moreover, the prion ([PSI⁺]) state of the endogenous S. cerevisiae Sup35p cannot be transmitted to the next generations via heterologous Sup35p or Sup35NM, originating from the distantly related yeast species Pichia methanolica. This suggests the existence of a ‘species barrier’ in yeast prion conversion. However, the chimeric Sup35p, containing the Sup35NM region of Pichia, can be turned into a prion in S. cerevisiae by overproduction of the identical Pichia Sup35NM. Therefore, the prion‐forming potential of Sup35NM is conserved in evolution. In the heterologous system, overproduction of Pichia Sup35p or Sup35NM induced formation of the prion form of S. cerevisiae Sup35p, albeit less efficiently than overproduction of the endogenous Sup35p. This implies that prion induction by protein overproduction does not require strict correspondence of the ‘inducer’ and ‘inducee’ sequences, and can overcome the ‘species barrier’.

To test the hypothesis that inaccurate DNA synthesis by mammalian DNA polymerase η (pol η) contributes to somatic hypermutation (SHM) of Ig genes, we measured the error specificity of mouse pol η during synthesis of each strand of a mouse Ig κ light chain transgene. We then compared the results to the base substitution specificity of SHM of this same gene in the mouse. Thein vitroandin vivobase substitution spectra shared a number of common features. A highly significant correlation was observed for overall substitutions at A-T pairs but not for substitutions at G-C pairs. Sixteen mutational hotspots at A-T pairs observedin vivowere also found in spectra generated by mouse pol ηin vitro. The correlation was strongest for errors made by pol η during synthesis of the non-transcribed strand, but it was also observed for synthesis of the transcribed strand. These facts, and the distribution of substitutions generatedin vivo, support the hypothesis that pol η contributes to SHM of Ig genes at A-T pairs via short patches of low fidelity DNA synthesis of both strands, but with a preference for the non-transcribed strand.

Despite extensive study, progress in elucidation of biological functions of amyloids and their role in pathology is largely restrained due to the lack of universal and reliable biochemical methods for their discovery. All biochemical methods developed so far allowed only identification of glutamine/asparagine-rich amyloid-forming proteins or proteins comprising amyloids that form large deposits. In this article we present a proteomic approach which may enable identification of a broad range of amyloid-forming proteins independently of specific features of their sequences or levels of expression. This approach is based on the isolation of protein fractions enriched with amyloid aggregates via sedimentation by ultracentrifugation in the presence of strong ionic detergents, such as sarkosyl or SDS. Sedimented proteins are then separated either by 2D difference gel electrophoresis or by SDS-PAGE, if they are insoluble in the buffer used for 2D difference gel electrophoresis, after which they are identified by mass-spectrometry. We validated this approach by detection of known yeast prions and mammalian proteins with established capacity for amyloid formation and also revealed yeast proteins forming detergent-insoluble aggregates in the presence of human huntingtin with expanded polyglutamine domain. Notably, with one exception, all these proteins contained glutamine/asparagine-rich stretches suggesting that their aggregates arose due to polymerization cross-seeding by human huntingtin. Importantly, though the approach was developed in a yeast model, it can easily be applied to any organism thus representing an efficient and universal tool for screening for amyloid proteins.

The concept of "protein-based inheritance" defines prions as epigenetic determinants that cause several heritable traits in eukaryotic microorganisms, such as Saccharomyces cerevisiae and Podospora anserina. Previously, we discovered a non-chromosomal factor, [NSI⁺], which possesses the main features of yeast prions, including cytoplasmic infectivity, reversible curability, dominance, and non-Mendelian inheritance in meiosis. This factor causes omnipotent suppression of nonsense mutations in strains of S. cerevisiae bearing a deleted or modified Sup35 N-terminal domain. In this work, we identified protein determinants of [NSI⁺] using an original method of proteomic screening for prions. The suppression of nonsense mutations in [NSI⁺] strains is determined by the interaction between [SWI⁺] and [PIN⁺] prions. Using genetic and biochemical methods, we showed that [SWI⁺] is the key determinant of this nonsense suppression, whereas [PIN⁺] does not cause nonsense suppression by itself but strongly enhances the effect of [SWI⁺]. We demonstrated that interaction of [SWI⁺] and [PIN⁺] causes inactivation of SUP45 gene that leads to nonsense suppression. Our data show that prion interactions may cause heritable traits in Saccharomyces cerevisiae. The data presented in the paper deepens and enriches the concept of protein-based inheritance. According to this concept, prion conformational switches change protein functional activity, and such changes are inherited. Here, for the first time, we demonstrate that heritable traits may appear not only due to a conformational switch of one protein but also can be caused by interactions between different prions. To identify the novel epigenetic factor that causes suppression of nonsense mutations in yeast, we applied our original method of proteomic screening of prions. We have shown that two yeast proteins, which normally do not interact, in prion form demonstrate genetic interaction: one is the key determinant of the suppression of nonsense mutation, while the second enhances this effect. Thus, by analogy with monogenic and polygenic inheritance, in the framework of the prion concept, we can distinguish “monoprionic” and “polyprionic” inheritance. We assume that new examples of polyprionic inheritance will be revealed using modern proteomic methods for identification of prions.

Amyloids are β-sheets-rich protein fibrils that cause neurodegenerative and other incurable human diseases affecting millions of people worldwide. However, a number of proteins is functional in the amyloid state in various organisms from bacteria to humans. Using an original proteomic approach, we identified a set of proteins forming amyloid-like aggregates in the brain of young healthy rats. One of them is the FXR1 protein, which is known to regulate memory and emotions. We showed that FXR1 clearly colocalizes in cortical neurons with amyloid-specific dyes Congo-Red, Thioflavines S and T. FXR1 extracted from brain by immunoprecipitation shows yellow-green birefringence after staining with Congo red. This protein forms in brain detergent-resistant amyloid oligomers and insoluble aggregates. RNA molecules that are colocalized with FXR1 in cortical neurons are insensitive to treatment with RNase A. All these data suggest that FXR1 functions in rat brain in amyloid form. The N-terminal amyloid-forming fragment of FXR1 is highly conserved across mammals. We assume that the FXR1 protein may be presented in amyloid form in brain of different species of mammals, including humans.