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.

Amyloids and their infectious subset, prions, represent fibrillary aggregates with regular structure. They are formed by proteins that are soluble in their normal state. In amyloid form, all or part of the polypeptide sequence of the protein is resistant to treatment with proteinase K (PK). Amyloids can have structural variants, which can be distinguished by the patterns of their digestion by PK. In this review, we describe and compare studies of the resistant cores of various amyloids from different organisms. These data provide insight into the fine structure of amyloids and their variants as well as raise interesting questions, such as those concerning the differences between amyloids obtained ex vivo and in vitro, as well as the manner in which folding of one region of the amyloid can affect other regions.

Fragmentation of amyloid polymers by the chaperone Hsp104 allows them to propagate as prions in yeast. The factors which determine the frequency of fragmentation are unclear, though it is often presumed to depend on the physical strength of prion polymers. Proteins with long polyglutamine stretches represent a tractable model for revealing sequence elements required for polymer fragmentation in yeast, since they form poorly fragmented amyloids. Here we show that interspersion of polyglutamine stretches with various amino acid residues differentially affects the in vivo formation and fragmentation of the respective amyloids. Aromatic residues tyrosine, tryptophan and phenylalanine strongly stimulated polymer fragmentation, leading to the appearance of oligomers as small as dimers. Alanine, methionine, cysteine, serine, threonine and histidine also enhanced fragmentation, while charged residues, proline, glycine and leucine inhibited polymerization. Our data indicate that fragmentation frequency primarily depends on the recognition of fragmentation-promoting residues by Hsp104 and/or its co-chaperones, rather than on the physical stability of polymers. This suggests that differential exposure of such residues to chaperones defines prion variant-specific differences in polymer fragmentation efficiency.

Cell death in response to distinct stimuli can manifest different morphological traits. It also depends on various cell death signaling pathways, extensively characterized in higher eukaryotes but less so in microorganisms. The study of cell death in yeast, and specifically Saccharomyces cerevisiae, can potentially be productive for understanding cell death, since numerous killing stimuli have been characterized for this organism. Here, we systematized the literature on external treatments that kill yeast, and which contains at least minimal data on cell death mechanisms. Data from 707 papers from the 7000 obtained using keyword searches were used to create a reference table for filtering types of cell death according to commonly assayed parameters. This table provides a resource for orientation within the literature; however, it also highlights that the common view of similarity between non-necrotic death in yeast and apoptosis in mammals has not provided sufficient progress to create a clear classification of cell death types. Differences in experimental setups also prevent direct comparison between different stimuli. Thus, side-by-side comparisons of various cell death-inducing stimuli under comparable conditions using existing and novel markers that can differentiate between types of cell death seem like a promising direction for future studies.

The yeast [PSI⁺] prion, formed by the Sup35 (eRF3) protein, has multiple structural variants differing in the strength of nonsense suppressor phenotype. Structure of [PSI⁺] and its variation are characterized poorly. Here, we mapped Sup35 amyloid cores of 26 [PSI⁺] ex vivo prions of different origin using proteinase K digestion and mass spectrometric identification of resistant peptides. In all [PSI⁺] variants the Sup35 amino acid residues 2–32 were fully resistant and the region up to residue 72 was partially resistant. Proteinase K-resistant structures were also found within regions 73–124, 125–153, and 154–221, but their presence differed between [PSI⁺] isolates. Two distinct digestion patterns were observed for region 2–72, which always correlated with the “strong” and “weak” [PSI⁺] nonsense suppressor phenotypes. Also, all [PSI⁺] with a weak pattern were eliminated by multicopy HSP104 gene and were not toxic when combined with multicopy SUP35. [PSI⁺] with a strong pattern showed opposite properties, being resistant to multicopy HSP104 and lethal with multicopy SUP35. Thus, Sup35 prion cores can be composed of up to four elements. [PSI⁺] variants can be divided into two classes reliably distinguishable basing on structure of the first element and the described assays.

Novel derivatives of Mycosidine (3,5-substituted thiazolidine-2,4-diones) are synthesized by Knoevenagel condensation and reactions of thiazolidines with chloroformates or halo-acetic acid esters. Furthermore, 5-Arylidene-2,4-thiazolidinediones and their 2-thioxo analogs containing halogen and hydroxy groups or di(benzyloxy) substituents in 5-benzylidene moiety are tested for antifungal activity in vitro. Some of the synthesized compounds exhibit high antifungal activity, both fungistatic and fungicidal, and lead to morphological changes in the Candida yeast cell wall. Based on the use of limited proteomic screening and toxicity analysis in mutants, we show that Mycosidine activity is associated with glucose transport. This suggests that this first-in-class antifungal drug has a novel mechanism of action that deserves further study.