No abstract

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 thrombin-catalyzed cleavage of N-terminal fibrinopeptide A (FPA) from the two Aalpha-chains of fibrinogen exposes aggregation sites with the critical sequence GPR located just behind FPA. It is well known that exposure of both GPR sites transforms fibrinogen into self-aggregating, fully coagulable alpha-fibrin monomers, but the fibrin precursor with one site exposed and one FPA intact has eluded description. The formation of this "alpha-profibrin" in the course of thrombin reactions and its distribution among both the aggregating and non-aggregating components of the reactions are characterized here by immunoprobing electrophoretic and gel chromatographic separations using monoclonal antibodies specific for FPA and for exposed GPR sites. These analyses show alpha-profibrin to be a non-aggregating derivative indistinguishable from fibrinogen in solutions that are rich in fibrinogen relative to dissolved fibrin. But alpha-profibrin forms soluble complexes with alpha-fibrin monomer under conditions in which it and fibrin predominate over fibrinogen. It was isolated as a complex with fibrin by gel chromatography of cryoprecipitates and then separated from the fibrin either by electrophoretic gel shifts induced with a peptide analog of the GPR aggregation site or by chromatographic gel shifts induced with monoclonal anti-FPA antibody. The weak aggregation of alpha-profibrin with itself and with fibrinogen conforms with prior indications that coupled interactions through the paired GPR sites on fibrin monomers are pivotal to their aggregation. It is suggested that alpha-profibrin may be a hypercoagulable fibrin precursor because it is converted to alpha-fibrin monomer faster than fibrinogen converts to monomer.

No abstract

In eukaryotes, termination of translation is controlled by polypeptide chain release factors eRF1 and eRF3, of which the former recognizes nonsense codons, while the latter interacts with eRF1 and stimulates polypeptide release from the ribosome in a GTP- dependent manner, and ABCE1, which facilitates ribosome recycling. In this work, we demonstrate that Pub1, a yeast protein known to be involved in stress granule formation, regulation of gene expression, and organization of the tubulin cytoskeleton, also plays a role in translation termination. Pub1 was shown to bind to ribosomes independent of eRF1 and eRF3 and to interact with the N-terminal glutamine-/asparagine-rich prion domain of eRF3 via its short C-terminal glutamine-rich tract. High velocity sedimentation in sucrose gradient demonstrated that Pub1 was preferentially associated with heavy polysomes enriched with terminating ribosomes. Lack of Pub1 decreased efficiency of nonsense readthrough at a majority but not all tetranucleotide stop signals. This distinguishes Pub1 from most other known binding partners of the release factors which were shown to modulate readthrough of all nonsense codons uniformly. The obtained data show that Pub1 can act as an accessory translation factor involved in fine-tuning of translation termination.

No abstract

Amyloids are fibrillar protein aggregates resulting from non-covalent autocatalytic polymerization of various structurally and functionally unrelated proteins. Previously we have selected DNA aptamers, which bind specifically to the in vitro assembled amyloid fibrils of the yeast prionogenic protein Sup35. Here we show that such DNA aptamers can be used to detect SDS-insoluble amyloid aggregates of the Sup35 protein, and of some other amyloidogenic proteins, including mouse PrP, formed in yeast cells. The obtained data suggest that these aggregates and the Sup35 amyloid fibrils assembled in vitro possess common conformational epitopes recognizable by aptamers. The described DNA aptamers may be used for detection of various amyloid aggregates in yeast and, presumably, other organisms.

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.